New genes encoding new proteolytic enzymes

FIELD: biotechnological methods.

SUBSTANCE: invention concerns new polynucleotides, which encode polypeptide having tripeptidylpentidase activity. To produce this polypeptide, host cell is transformed with polynucleotide or polynucleotide-containing vector and then cultured under suitable polynucleotide expression conditions.

EFFECT: enabled production of new protease from thread fungi.

14 cl, 3 tbl, 11 ex

 

The scope to which the invention relates.

The present invention relates to a new identified polynucleotide sequences comprising genes encoding new protease isolated from Aspergillus niger. The present invention relates to a full-sized nucleotide sequence of these new genes, cDNA sequences, including these full coding sequences of new proteases, as well as to amino acid sequences of the full-functional proteins, as well as their fragments and variants. The present invention also relates to methods of using these enzymes in industry and to methods of diagnosing fungal infections. The present invention also relates to cells, transformed polynucleotide of the present invention to cells in which the protease of the present invention is genetically modified to increase or decrease its activity and/or expression level.

Prior art

Proteolytic enzymes

Proteins can be considered as heteropolymer, which consist of amino acid structural elements connected by a peptide bond. A recurring element in proteins is the Central atom of the alpha carbon amino and carboxyl group. The so-called the I side chain of amino acids, except for glycine, replaces one of the two remaining hydrogen atoms at the alpha carbon. Side chain of amino acids makes the Central atom of the alpha-carbon asymmetric. In the composition of proteins, mainly found L-enantiomers of amino acids. Different types of polymerized amino acids define the following terms. "Peptides are short chains of amino acid residues in a certain sequence. Although, actually, you cannot specify precisely the maximum number of residues, however, this term usually refers to a chain that has properties defined, mainly, its amino acid composition, and which does not have a fixed three-dimensional conformation. The term "polypeptide" is usually used to refer to longer chains, which usually has a specific sequence and length, which, in principle, is sufficient for the formation of three-dimensional structures. The term "protein" usually means polypeptides that are found in nature and has a specific three-dimensional structure. If the main function of protein is the catalysis of a chemical reaction, typically, such proteins are called enzymes. By proteases called enzymes, which catalyze the hydrolysis of peptide bonds in the (poly)peptides and proteins.

Under physiological conditions proteases catalyze hydro is of the peptide bond. The international Union of biochemistry and molecular biology (1984) recommends the use of the term "peptidase" for sub-groups of the peptide bond hydrolases (subclass ES). The terms "protease" and "peptidoglycans" are synonymous with "peptidase" and can also be used in the present description. Proteases include two classes of enzymes: endopeptidase and ectopeptidases, which split peptide bonds at specific points in the protein and sequentially removing amino acids from either the N-or C-end, respectively. The term "proteinase" is used synonymously with the term "endopeptidase". The peptide bond can be present in the context of di-, tri-, tetrapeptides, peptides, polypeptides or proteins. Basically, the amino acid compositions of natural peptides and polypeptides include 20 different amino acids have L-configuration (with the exception of glycine, which has no chiral center). However, the proteolytic activity of proteases is not limited to peptides, which contain only 20 natural amino acids. Can also be cleaved peptide bond between the so-called non-natural amino acids and the peptide bond between the modified amino acids or amino acid analogues. Some proteases recognize the D-enantiomers of amino acids in certain positions. Mostly significant with areaselector proteases is a very important factor, which allows their use in methods of chemical separation. Many proteases have other interest activities, such as esterna activity, telestrada activity and (de)Amidala activity. These auxiliary activity usually are not limited to amino acids and can be successfully used in reactions of biological transformations in the field of fine chemical synthesis.

Of particular interest to the protease filamentous fungi, eukaryotic microorganisms, for several reasons. It is obvious that the basic process of hydrolytic cleavage of peptide bonds in proteins is costly, and if it is not appropriately regulated, can have a negative effect on the microorganism. The desired limit proteolytic activity can be achieved through the specificity of proteases; the compartmentalization of enzymes and substrates in the cell; modification of substrates recognized by the appropriate protease, regulation through activation of imagenow and by the presence or absence of specific inhibitors, as well as by regulating gene expression of the protease. In fungal proteases are also involved in other fundamental cellular processes, including metabolism intracellular protein processing, translocation, sporule the Oia, development and differentiation. Indeed, Aspergillus nidulans and Neurospora crassa were used as a micro-model for the analysis of the molecular basis of a number of physiological processes and development processes. Their genetics to provide direct access to biochemical and genetic studies in certain conditions, nutrition and cultivation. In addition, he was selected a large group of fungi pathogenic for humans, cattle and crops, and it has been suggested that in their pathogenicity (penetration into the host organism, counter-defense mechanisms of the host and/or infection in the process of feeding) the role of proteolysis. Proteases are also often used in laboratory, clinical and industrial methods; both microbial and nemikrobnoy proteases are widely used in the food industry (baking, brewing, production of cheese and tenderizes meat), in the leather industry and in the industrial production of biological detergents (Aunstrup, 1980). Recently, in connection with the industrial application of some filamentous fungi, in particular Aspergillus, as hosts for the production of homologous and heterologous proteins, again increased interest in fungal proteases (van Brunt, 1986ab). Protease is often difficult heterologous exp is essay and the homologous overexpression of proteins in fungi. In particular, the heterologous expression of a negative effect of proteolytic cleavage products of the expression of homologous proteases. Specified commercial interest was the impetus for a detailed study of the spectrum proteolytic proteases and design deficient in protease strains, which resulted in gaining more knowledge about the expression of proteases and their regulation in these organisms. Therefore, at the present time, there is a need for identification and elimination of new proteases in filamentous fungi.

Microorganisms, such as fungi, are particularly suitable for large-scale production of proteins, and especially if such proteins are secreted into the environment. In these processes of production play a role of proteolytic enzymes. On the one hand, specific proteolytic enzymes, mainly required for correct processing of the protein target and a favorable exercise of the metabolic functions of the host-producer. On the other hand, proteolytic cleavage can significantly reduce the release of secreted proteins. Improper installation in the path secretion can lead to cleavage by intracellular proteases. In this regard, when the production of heterologous proteins, can be difficult. The exact mechanisms of proteoliticheskikh processes, responsible for the metabolism of protein, which deviate from the secretory pathway in fungi, is not yet entirely clear. In eukaryotes, the degradation of cellular proteins is achieved using the proteasome and is usually done by tagging degradable protein ubication. In fungi, proteosome and vacuole-type proteases are likely candidates for proteolytic cleavage badly laid secretory proteins. Proteolytic degradation is probably the cytoplasmic, but we cannot exclude the existence of proteases in the endoplasmic reticulum. From the viewpoint of improvement of the master's producer strain proteolytic system may be an interesting target for genetic improvement of design and production strains. Additional copies of the genes of proteases, overexpression of certain proteases, modification of transcriptional regulation, as well as "knock-out" procedure for deletions of genes of proteases will allow a better understanding of the function of this protease. Deletion of the genes encoding the protease, can serve as a valuable strategy for modification of the strain of the host is performed in order to increase the yield of production of homologous and heterologous proteins.

Eukaryotic microbial proteases have been described North (1982). Recently, Suarez Rendueles and Wolf (1988) described Protea the s S. cerevisiae and their functions.

In addition to hydrolytic cleavage ties, proteases may also be involved in binding. In this aspect, the term "communication" includes not only the peptide and amide linkages, but also and ester bonds. The ability of proteases to catalyze the cleavage or formation of a specific connection in the first place, depends on thermodynamics of the reaction. Such an enzyme as protease, does not affect the equilibrium of the reaction. The equilibrium of this reaction depends on the specific conditions under which this reaction is carried out. In physiological conditions, thermodynamics of these reactions may be favorable for hydrolysis of the peptide due to the high degree of thermodynamically stable structure zwitterionic product. Based on the physico-chemical laws affecting the equilibrium of the reaction, or by changing the concentration or nature of the reactants and products, or picking up the kinetic parameters of the enzymatic reaction, can be used protease for the synthesis of peptide bonds. Adding mixed with water, organic solvents leads to the decrease of the degree of ionization of the carboxylic component and, thereby, to increase the concentration of substrate available for the reaction. To increase the output often use a two-phase system, water mimetics, reversed micelles, anhydrous environment or Modific rowanne amino and carboxyl groups, stimulating the precipitation products. If available protease with appropriate properties, then the use of proteases for the synthesis would give significant benefits. Since proteases are stereoselective and regioselective, sensitive groups on the reactants are usually not in need of protection and these reagents do not have to be optically pure. If the conditions of the enzymatic synthesis are soft, you can avoid racemization and decomposition of labile reagents or products. In addition, linkages between amino acids, suitably selected protease can also bind other compounds having a primary amino group, thiol group or carboxyl group. In addition, some proteases can be synthesized esters, thiol esters and amides. It has been shown that proteases have regiospecificity in the acylation of mono-, di - and trisaccharides, nucleosides and Riboflavin. The problems associated with stability, which sometimes occur when the hard reaction conditions, can be solved by a proper choice of reagents. Encapsulation and immobilization not only stabilize the enzymes, but also make it easy to separate and isolate them from the reaction medium. The density of crosslinks, treatment with aldehydes or floor surface is t some polymers, such as dextrans, polyethylene glycol and polyimide, can significantly increase the service life of the biocatalyst.

The natural role of proteases

Traditionally, the protease was considered proteolytic enzymes that can break down proteins into small peptides and/or amino acids, and the role of which consists in the hydrolysis of food proteins or involved in the metabolism of cellular proteins. In addition, it was shown that proteases also play a key role in cellular processes a wide range in accordance with the mechanisms of selective modification by limited proteolysis, and therefore they have important regulatory functions (Holzer & Tschensche 1979; Holzer & Heinrich, 1980). It is assumed that the specificity of the protease closely related to its physiological function and type of its expression. As for function-specific protease, its localization in most cases is very important; for example, series and using periplasmatic proteases involved in the degradation of the protein, whereas many membrane-associated proteases play an important role in protein processing (Suarez Rendueles & Wolf, 1988). The different roles of proteases in many cellular processes can be subdivided into four main functions of proteases: 1) degradation of the protein, 2) post-translational processing and (in)activation of specific proteins, 3) morphogenesis and pathogenesis.

The obvious role of proteases in organisms that use protein as a nutrient source, is the hydrolysis of nutrients. In fungi, this should lead to their degradation outside the cells under the action of extracellular proteases with broad specificity. Degradation of protein is also important for the rapid metabolism of cellular proteins and allows you to remove unnecessary cells proteins and adapt their complement protein to changing physiological conditions. Usually protease with broad specificity should be very well regulated to protect cellular proteins, non-protein-targets from random degradation.

In contrast to hydrolysis synthesis of polypeptides occurs in vivo through ATP-driven process on the ribosome. Ultimately, the sequence in which amino acids are linked, is determined from the information provided by the genome. This process is known as transcription. Primary products broadcast, usually are longer than the final functional foods, and after transcription is usually necessary processing such protein precursor by the action of proteases. Proteases play a key role in the maturation of these proteins precursor and formation of the final functional protein. In contrast to the high degree adjustable "cuts" and "reverse engineering" of protein protease can have a highly destructive effect and can lead to complete degradation of polypeptides on peptides and amino acids. To prevent this, free of proteolytic activity, before it is required, the protease should be subject to intense regulation. Many proteases are synthesized as larger precursors, known as Imogene that, if necessary, become activated. It should be noted that such activation always occurs by proteolysis. In addition to direct involvement in processing, selective activation and inactivation of individual proteins are well-known processes catalyzed by specific proteases.

It is obvious that the selectivity of limited proteolysis almost always occurs in the interaction of the protease-substrate. This specificity can be provided by a proteolytic enzyme that recognizes a specific amino acid target sequence. On the other hand, it can also be due to a selective effect on the "site processing" under certain conditions, such as pH, ionic strength or secondary modification that allows nonspecific proteases, one way or another, to catalyze highly specific event. An example of an event of this type is the activation using imagenow with limited proteolysis.

Morphogenesis or differentiation can be defined as forming the series of events, leading to the transition from one state to the body in another state. Although, in many cases, a direct correlation between proteases and morphological effects has not been established, however, there are data that suggest a significant role of proteases in the morphogenesis of fungi, that is, in addition to observed the intensive metabolism of protein in the process of differentiation, sporulation and germination of spores, protease, obviously, are directly involved in normal processes such as branching tips of the hyphae and the formation of partitions (Deshpande, 1992).

It is believed that the species of Aspergillus, in particular, A.fumigatus and A.flavus are the etiological factors of diseases in humans and animals, called aspergillosis (Bodey & Vartivarian, 1989). And again, an assumption was made that proteases play a role in virulence A.fumigatus and A.flavus, just as it was observed in many studies on the relationship secreted protease and virulence of bacteria. Indeed, the majority of human infections caused by Aspergillus species, are characterized by intense degradation of lung parenchyma, which consists mainly of collagen and elastin (Campbell et al., 1994). Research has focused on the study assumed the role of secreted proteases in virulence A.fumigatus and A.flavus, which are heads of the diversified human pathogens and, as you know, have latinalicious and collagen activities (Kolattukudy et al., 1993). It was shown that these latinalicious activity correlate in vitro with infectivity mice (Kothary et al., 1984). It is known that two secreted protease, alkaline serine protease (ALP) and neutral metalloprotease (MEASURES)are produced A.fumigatus and A.flavus. Both genes encoding these proteases of A.fumigatus, were isolated, characterized and subjected disruptive (Reicherd et al., 1990; Tang et al., 1992, 1993; Jaton-Ogay et al., 1994). However, it was shown that double alp-mep-mutants in their pathogenicity not differ from wild-type strains. Therefore, it was concluded that the secreted protease A.fumigatus identified in vitro, are not the main factors for invasion in the fabric (Jaton Ogay et al., 1994). Although A.fumigatus responsible for the formation of only a small part of the dispute airborne mold, this fungus most often stands out from the lungs and sputum (Schmitt et al., 1991). Another explanation virulence of fungi may be the fact that conditions in the bronchi (temperature and nutrient medium) are more favorable for parasite growth A.fumigatus. Therefore, if the protection of the host from pathogens weakened by the introduction of immunosuppressive drugs or diseases such as AIDS, invasive aspergillosis may be caused by environmental factors. There are four main the class of proteases, which are the main functional groups in their active site, namely, "serine", "thiol" or "cysteine", "aspartic" or "carboxyl" protease and metalloprotease". A detailed description of these large and small classes of proteases, and unclassified proteases can be found in "Methods in Enzymology", part 244 and 248 (A.J.Barrett ed., 1994 and 1995).

The specificity of proteases

In addition to the catalytic mechanism of action of protease, another important aspect of proteolytic enzymes is the specificity of proteases. The specificity of the protease indicates the specific substrates of proteases, which, in all probability, hydrolyzes this protease. Twenty natural amino acids provide ample opportunities for the design of peptides. For example, using the twenty amino acids can be constructed about 400 dipeptides and 800 different tripeptides and the like, using longer peptides, the number of possible structures becomes almost unlimited. Some protease hydrolyzing only specific sequences highly specific position. The interaction of the protease with a peptide substrate may include from one to ten amino acid residues of the peptide substrate. In the case of large protein substrates to interact with proteases may even larger number of residues of the subst the ATA. However, this probably leads to less specific interactions with protease residues outside the binding pocket of the active site. Basically, specific recognition is limited to a linear peptide that binds in the active site of the protease.

The nomenclature for describing the interactions of the substrate with a protease was developed in 1967 Schechter & Berger (Biochem. Biophys. Res. Com. 1967, 27, 157-162) and is currently widely used in the literature. It is believed that this system of amino acid residues of the polypeptide substrate in contact with the so-called subsites in the active site. For convenience, these subsites on the protease were designated S (for subsites), and the corresponding amino acid residues were identified P (for peptide). Amino acid residues from the N-terminal side of fissile relations were marked P3, P2, P1, and residues from the C-terminal side were designated P1', P2', P3'. The remains of the P1 or P1' represents amino acid residues located near fissile communication. The remains of the substrate located near the site of cleavage can then be numbered up to P8. The corresponding subsites of this protease, which will complementary remains that communicates with the substrate, were numbered S3, S2, S1, S1', S2', S3', etc. Preference subsites in the binding site with peptidomimetics preference protease in the cleavage of certain specific amino acid sequences in a particular place. Amino acid sequence of the substrate must conform to the preference shown by the subsites. The specificity with respect to a specific substrate clearly depends on the affinity of binding with the substrate and the speed at which the cleaved bond is hydrolyzed. Therefore, the specificity of the protease to a specific substrate typically is determined by the ratio kcat/Km, which is more known under the term "specificity constant". In the specificity constant, kcat mean metabolic rate, and Km denotes the dissociation constant.

In addition to the amino acid residues involved in catalysis and binding, protease contain many other basic amino acid residues. Some remnants are important for laying, some remnants support the entire three-dimensional structure of the protease, some residues may participate in the regulation of proteolytic activity, and some remnants can target the protease at the specific site. Many of them contain, outside the active site, one or more binding sites with metal ions. These metal ions, in most cases, play a role in stabilizing the structure. In addition, secreted eukaryotic microbial protease can be highly glycosylated. This can occur both N-and O-linked glycosylation. Glycosylation may contribute to the laying of the protein, can increase the solubility and to prevent aggregation and, thus, to stabilize the Mature protein. In addition, the glycosylation may affect the secretion and binding of water under the action of the protein.

Regulation of the proteolytic activity

In order to avoid unwanted proteolytic cleavage of a large number of proteases are exposed to intense regulation of proteolytic activity. To some extent this regulation occurs at the transcriptional level. So, for example, fungi gene transcription secreted protease is obviously sensitive to external carbon and nitrogen sources, whereas genes encoding intracellular proteases, are not sensitive to such sources. Fungi are sensitive to extracellular pH, and some genes are regulated by pH. In the process of transcriptional regulatory proteins play a crucial role. Proteolytic processing of such regulatory proteins often serves as a "switch", or "includes"or "disables" regulatory proteins.

Protease undergo intramolecular and intermolecular regulation. This means that some amino acids in the molecule proteolytic enzyme plays a major role in this regulation. The FR is SHL usually are synthesized as larger precursors, known as Imogene, which are catalytically inactive. Typically, the elongation of the peptide chain, which makes the protease-inactive precursor, is localized at linekona of this protease. This predecessor is better known as a Pro-protein. Since many proteases, versions in this way, are secreted from cells, they also contain a signal sequence (pre-sequence) to a full-sized predecessor, was synthesized as pre-Pro-protein. Besides the fact that this protease is inactive, this Pro-peptide often plays an important role in mediating styling product. Examples of proteases are serine proteases (allpolitics protease, subtilisin, equality, prohormone-convertase), thiol protease (cathepsin L and crusian), aspartic protease (proteinase a and cathepsin D) and metalloprotease. In addition, the Pro-peptide may play a role in cellular transport, either individually or in combination with signal peptides. It can facilitate interaction with cellular chaperones, or it can facilitate the transport through the membrane. The size of the extension section in the pre-Pro-protein-precursor can vary greatly, from short peptide fragment to the polypeptide, and this site can exist as Autonomous units of the installation. In particular, as observed in most cases, these larger extension parcels are strong protease inhibitors even after removal from the protease. It has been observed that even after removal of such Pro-peptides can facilitate proper stacking of the protease. It can be assumed that such Pro-peptides act as molecular chaperones and individual or collective co-expression of these Pro-peptides may be preferred for the production of protease.

Levels of regulation of proteases that are secreted into the environment, and proteases, which remain inside the cell, there is a significant difference. Usually, after activation, protease, secreted into the environment, no longer subject to the regulation, and therefore they have a relatively simple molecular structure, consisting of a single globular module. Intracellular protease should be subject to continuous regulation in order to avoid cell damage. Unlike imagenow secreted proteases, in a more complex regulatory proteases, between the signal sequence and an activation domain of Imogene proteolytic module can be entered very large polypeptide segments. The study of the structure and function indicates that such deproteinize part can participate in interactions with macroscopic structure of the AMI, membranes, cofactors, substrates, effectors, inhibitors and ions that regulate the activity and activation of proteolytic(s) module(s) or his (their) Imogene(s). Such nefrotoksicheskoe modules vary greatly in their sizes and structures. Many of these modules can exist independently of proteolytic module. So we can assume that these modules do not depend on the structural and functional units that are Autonomous in relation to styling. The value of such modular organization is that the acquisition of new modules may confer protease-recipient new binding specificity and can lead to sharp changes in its activity, regulation and targeting. The modular organization of proteolytic enzymes can also be used in methods of molecular biology to provide new interactions, regulation, specificity and/or targeting by "shuffling" of modules. Though, mostly, these additional modules are present as N - or C-terminal extension, however, there was also a large insertion in the outer loops of the catalytic domain. Obviously, in this case also the main laying protease is a basic topology for the formation of functional proteolytic molecules and that this insert can R smotriatsa as substructure, laid on the surface of proteolytic module. Molecular structure

In principle, the main topic of discussion is the modular organization of the larger natural proteins. In particular, in larger multi-module frames, typical proteolytic modules have dimensions of, on average, from 100 to 400 amino acids. This corresponds to the average size of most globular proteolytic enzymes, which are secreted into the environment. As discussed above, the polypeptide modules are polypeptide fragments, which can be stacked and can function as independent molecules. Another name for such modules are "domains". However, the term "domain" is used in a broader context than the module. Used herein, the term "domain" usually means a portion of the polypeptide chain, which has a typical topology stacking in three-dimensional structure. In the protein domains interact at different levels, but less intensively than structural elements within the domains. The literature also uses other terms, such as the subdomain and unit installation. In fact, it has been observed that many proteins that have the same specific functional groups can have the same domains. Such domains can be identified by their primary structure, which may have a sequence of ODA is divided type, which is typical for this domain. Typical examples are the mononucleotide-binding fold; the domains that communicates with the pulp; the motif of DNA binding "helix-turn-helix; domain zinc fingers; EF-shoulders; membrane anchors. The term "module" refers to the domains that are presumed to be capable of Autonomous styling and function. Specialist known methods identification of specific domains in the primary structure using a publicly available computer programs for the specified structure and homologous sequences derived from other organisms or species.

Although multimodal or multi-domain proteins can be a chain of type "string of beads", however, was observed significantly more complex architecture. If on the same polypeptide chain are different "beads", these "beads" typically referred to as modules or domains. If the "beads" are not present on the same polypeptide chain, but form an ordered structure through non-covalent interactions, in this case, use the term "subunit". Subunit can be transcribed in the same gene or different genes. After transcription, multimodal protein can undergo proteolytic processing, resulting in the formation of the sets of the subunits. A separate subunit may consist of multiple domains. Basically, the smaller globular proteins containing 100-300 amino acids, usually consist of only one domain.

Molecular classification of proteolytic enzymes

Basically, proteases are classified according to their molecular properties and functional properties. Molecular classification based on the primary structure of the protease. Primary structure of a protein is its amino acid sequence, which may occur from the nucleotide sequence of the corresponding gene. Intensive studies on the affinity of the primary structures, can lead to the identification of similarities catalytic mechanism and other properties that can even relate to functional properties. Used herein, the term "family" means a group of proteases that reveal the evolutionary relationship established on the basis of similarity in their primary structures. It is obvious that the members of this family are descended from the same ancestor, but then underwent divergence during evolution. In addition, within the family, these proteases are divided into subgroups based on their primary structures that have been installed by more detailed clarification of their sequence on the basis of results of comparison of p is Samast. Classification of three-dimensional stacking of proteases may include secondary structure, tertiary structure and Quaternary structure. In General, the classification of secondary structure is limited to the content and approximate orientation of secondary structure elements. The similarity of the tertiary structures allows to identify superfamily, or clans. The superfamily or a clan is a group of families who, obviously, have a common "ancestor", because they reveal a similar 3-dimensional styling. Basically, the three-dimensional structure is more conserved than primary structure. Therefore, the similarity of primary structure does not always reflect similar functional properties. Indeed, functional properties can change dramatically, which can lead to the emergence of new interest properties. Currently, the Quaternary structure is not used for classification of different proteases. This may be due to some displacement of the structural databases in simple globular proteases. Many proteolytic systems that undergo activation, regulation or complex cascade of reactions probably consist of multiple domains or subunits. The General trends of the structural organization of such protease systems Pref is going to new types of classification.

Classification in accordance with the specificity

In the absence of sequence information proteases, they can be classified in various types of functions. Classification and names of the enzymes in accordance with the reactions that they catalyze, is the main principle in the nomenclature of enzymes. This approach is also based on the principle of EU-numbering of enzymes (Enzyme Nomenclature 1992, Academic Press, Orlando). Two types of proteases (EC 3.4) can be determined in accordance with the nomenclature of enzymes Enzyme Nomenclature 1992, i.e. ectopeptidases (EC 3.4.11-19) and endopeptidases (EC 3.4.21-24, 3.4.99). Endopeptidase break down the peptide bonds at internal sites of the peptide chain, which are far from its end. Ectopeptidases otscheplaut residues only from the ends of the peptide chain. Ectopeptidases acting at the free N-Terminus, can release one amino acid residue, dipeptide or Tripeptide and are called, respectively, aminopeptidase (EC 3.4.11), dipeptidylpeptidase (EC 3.4.14) and tripeptidylpeptidase (EU 3.3.14). Protease that initiates the processing of the peptide from the carboxyl end, resulting in the release of one amino acid, called carboxypeptidases (EC 3.4.16-18). Patibilities (EC 3.4.15) otscheplaut dipeptide from the carboxyl end. Exo - and endopeptidase, in General, represent dipeptidase (EC 3.4.13), which specification is epicski split only dipeptides in their two amino acid halves. Omega peptidases (EC 3.4.19) remove terminal residues, which are substituted, cyclic or attached isopeptide connections.

In addition to the position in which the protease cleaves the peptide chain, for each type of proteases and possibly more separation in accordance with the nature of the preferred amino acid residues in the substrate. Basically, proteases can be classified into a wide, medium and narrow specificity. Some protease just call for specific proteins or polypeptides, which they hydrolyzing, for example keratinase, collagenase, elastase. Narrow specificity may be limited to one specific amino acid or one specific sequence that can be removed or dissolved, respectively. If protease reveals a special preference for one amino acid in the P1 or P1'-position, name this amino acid may be its determinant. For example, polyaminoacids removes the Proline of aminocore peptide (Proline represents the balance of P1). X-Pro or Proline are used if the split link on the sidebar eminiai this Proline (Proline represents the balance of P1'), for example, prolylcarboxypeptidase removes the Proline at carboxilic. Polyangiitis (or Pro-X) splits in position behind the Strait is on, and prolinamide (X-Pro) splits in position ahead of Proline. Amino acid residue in front of cleaved peptide bond, means the amino acid residue that enters the carboxyl group in the peptide bond. Amino acid residue behind cleaved peptide bond, means the amino acid residue that enters the amino group in the peptide bond. According to common agreement amino acid chain extends from aminobenzo (start) to carboxylic (end), and accordingly carry out the numbering of the amino acids. Endoprotease can also have a clear preference for a particular amino acid in position P1 or P1', such as glycolaldehyde, peptidyltransferase, glutamyltranspeptidase. In addition, the protease may have a preference for a particular group of amino acids, which have some similarity. This preferred group of amino acids may include hydrophobic amino acids, only bulk hydrophobic amino acids, a small hydrophobic amino acid or a small amino acids, a large positively charged amino acids, etc. in Addition to preferences for residues P1 and P1', there may also be special or exclusive preference to the remains, preferred other subsites this protease. This is Dagestana preference may result in that proteases are highly specific to sequences that satisfy the many requirements for binding. Generally speaking, it should be noted that proteases are rather non-specific enzymes. Even very specific protease can cleave peptides, which are not the object is usually observed preferences protease. In addition, it should be noted that on the preferences of proteases can influence ambient conditions, such as pH, temperature, ionic strength, water activity, presence of solvents, the presence of competing substrates or inhibitors. Environmental conditions can affect not only the protease, but also on the path representation of the protein substrate by the protease.

Classification on the catalytic mechanism

Proteases can be classified based on their catalytic mechanisms. It should be noted that for each of the catalytic mechanism of the above classification of proteases based on their specificity, leading to the subsequent division of the proteases according to their mechanism of action. There are four major classes of proteases, which were named according to their main functional groups in the active site, namely serine protease (EC 3.4.21 endopeptidase, EC 3.4.16 the carboxypeptidase), thiol or cysteine proteases (EC 3.4.22 andhope tigase, EU 3.4.18 the carboxypeptidase), carboxyl or aspartic proteases (EC 3.4.23 endopeptidase) and metalloprotease (EC 3.4.24 endopeptidase, EC 3.4.18 the carboxypeptidase). For members of each of the catalytic types of proteases are characterized inhibitors. These small reversible inhibitors modify amino acid residue of the active site of the protease. For example, the serine protease is inactivated by phenylmethylsulfonyl (PMSF) and diisopropylfluorophosphate (DFP), which reacts with the active serine, whereas chloromethylketone derivatives react with histidine of the catalytic triad. Phosphoramidon and 1,10-phenanthrolin usually inhibit metalloprotease. Inhibition by pepstatin usually indicates aspartic protease. A specifically inhibits thiol protease. Lactatin and bestatin inhibit various amino peptidases. While there are significant differences in the protease susceptibility to inhibitors, even within a single catalytic class. To some extent this may be due to the specificity of the protease. When the architecture of the binding site prevents the mechanism, according to which the inhibitor affects the catalytic site, this protease "escapes" from inhibition, and identification of the type of mechanism of inhibition could not be implemented. So, for example, the R, hemostatis is a strong inhibitor of serine proteases with specificity that is similar to the specificity of chymotrypsin. Lactational inhibits anastasopoulou serine protease and does not react with trypsin or chymotrypsin; 4-amido-PMSF (APMSF) only inhibits serine protease with a specificity similar to trypsin. An extensive discussion of the use of inhibitors in the classification of proteases found in the work of Barret and Salvesen, Proteinase Inhibitors, Elsevier Amstardam, 1986; Bond and Beynon (eds), Proteolytic Enzymes, A Practical Approach, IRL Press, Oxford, 1989; Methods in Enzymology, eds E.J.Barret, volume 244, 1994 and volume 248, 1995; E.Shaw, Cysteinyl proteinases and their selective inactivation, Adv Enzymol. 63:271-347 (1990)Klassifikaciya on the optimal operating conditions

The catalytic mechanism of action of proteases and requirements to their conformational integrity determined, mainly, the conditions under which data can be used protease. The determination of the optimum conditions of application of the protease is a major challenge. Often the conditions under which the protease must operate, are not optimal and need to find a compromise between the ideal conditions for this particular application and conditions that should be most suitable for this protease. In addition to the specific properties of the protease, it should also be noted that the presentation of protein substrates depends on the conditions, and, in fact, it is also what determines the terms, which are the most effective for proteolysis. The parameters of the enzyme suitable for use are, for example, dependence on pH, temperature dependence, sensitivity to metal ions or dependence on metal ions, ionic strength, salt concentration, and compatibility with the solvent. The other most important factor is the specific activity of the protease. The greater specific activity of this enzyme has, the less he needs a specific conversion. The lower requirements to the enzyme, the lower its cost and lower levels of protein impurities.

pH is a key parameter that determines the functioning of the protease used for certain purposes. Therefore, the dependence on pH is an important parameter for a group of proteases. Major groups are usually divided protease is an acid protease, neutral protease, alkaline protease and highly alkaline protease. The optimum pH corresponds to a proteolytic mechanism only to a certain extent, for example, in most cases, the aspartic protease operates optimally only at acid pH; metalloprotease and thiol protease optimally operate from approximately neutral to slightly alkaline pH; serine proteases are active mainly in alkaline the high-alkaline environment. For each class of proteases known exceptions. In addition, the role of water activity in the system. The optimum pH for the protease is defined as the range of pH at which this prosthesis has an optimal rate of hydrolysis for most of its substrates in the environment and in specific conditions. This range may be low, for example, in one pH unit, and is wide enough for 3-4 units. Basically, the optimum pH also depends on the nature of the protein substrate. The speed of the functional cycle of the protease and its specificity can vary depending on pH. For some purposes it may be desirable to use a protease at pH values which are far from optimal, for example, in the case when it is necessary to avoid the production of less desirable peptides. Such less desirable peptides can be, for example, very short peptides or peptides, which gives a bitter taste. In addition, a narrower specificity can serve as the basis for selection of conditions that do not correspond to the optimal conditions in terms of speed functional cycle. Depending on the pH of the specificity of the protease can be narrow, for example, it can cleave the peptide chain only in one particular position in front of or behind a particular amino acid, or it may the be wider, for example, when the protease cleaves the chain in many provisions, either in front of or behind amino acids of many types. Indeed, the dependence on pH can be an important tool for the regulation of proteolytic activity in a certain order. When the pH changes during proteolysis, such proteolysis may cease spontaneously and does not require additional processing to inactivate the protease. In some cases, the proteolysis may be a regulator of pH changes.

When using proteases, a very important factor is their technological and functional stability. As the stability of the prosthesis largely depends on the operating temperature, this parameter is often referred to as thermal stability. Generally speaking, the stability of the protease indicates the retention of the proteolytic activity of proteases in these particular circumstances. Such specific terms may include conditions for fermentation conditions during the selection and subsequent processing of the enzyme, storage conditions, conditions of preparation and functioning of the enzyme or the conditions of its application. If specific conditions include stability at elevated temperatures, such stability, basically, is called stability. Besides the well-known causes inactivation of the enzyme,such as chemical modification, the deployment of a molecule of the enzyme, its aggregation and the like, the main problem is that these enzymes are easily autodegradation. Therefore, when using proteases, the optimum temperature is an important parameter for a group of proteases. Although there are various definitions, however, from an economic point of view, the most frequent determination of the optimal temperature is the temperature at which the protease is the most productive for this purpose. The productivity of protease depends on the stability and the speed of its functional cycle. While elevated temperature, generally, increases the speed of the cycle, rapid inactivation leads to a decrease in the rate cycle and, ultimately, to low productivity. Conformational stability of protease in the process will determine its maximum operating temperature. The temperature at which this protease loses its active conformation, often referred to as a deployment point or melting point, and can be determined by various methods, for example, using NMR, spectroscopy, circular dichroism, differential scanning calorimetry, etc. Deployment molecule protease is usually accompanied by a sharp increase in the rate of its autodegradation.

In cases when the required low temperature, selecting protease should focus on its high activity at low or moderate temperatures. Since under these conditions the inactivation of protease occurs relatively slowly, then the activity under these conditions may largely determine its productivity. In processes where the activity of the protease is only required in a very short period of time, the stability of the protease can be used to "enable" or "disable" protease. In this case, instead of the highly thermostable protease may be preferable to use more labile protease.

Other environmental parameters that may play a role in the selection of a suitable protease, may be its sensitivity to salts. Compatible with ions of metals that are often present in low concentrations in various natural substances, may be crucial for some applications. As metalloprotease, some ions can replace the catalytic metal ion and reduce or even completely suppress the activity of the protease. In some applications, to prevent leakage of metal ions coordinated with the protease, it is necessary to add these metal ions. It is well known that to maintain the stability of the enzyme and saved what I term you need to add calcium ions in order to prevent the dissociation of calcium, associated with the protein.

Most microorganisms find a certain tolerance and adapt to changes in environmental conditions. Therefore, as was shown on the basis of proteolytic spectrum of such a microorganism, it is able to produce at least the same tolerance. Such proteolytic range can be covered by many proteases, which together cover the "window" of the spectrum, or it can be covered by several proteases wide range. If we take into account the whole range of proteolytic microorganism, it is important may be localization.

Cellular localization and characterization of proteolytic processing and degradation

From the point of view of industrial applications, protease, which excretiruyutza of cells, have specific advantages in terms of large-scale productivity and tolerance to stress, because they are viable in the absence of cellular protection. A large group of cellular proteases can be also subdivided into soluble and membrane-associated protease. A group of membrane-associated proteases may include a protease associated with a membrane with both internal and external parties. Intracellular soluble protease can be, in addition, assistance is Helen in accordance with specific compartments of the cell, in which they are present. Since the cell, to some extent, protects protease from the external environment and as a cell regulates existing conditions, the intracellular protease may be more sensitive to significant changes in environmental conditions, and, in this case, the optimal parameters can be better correlated with the specific conditions within the cell. Knowledge of the conditions of cell compartment which contains the protease, may indicate its preference. If extracellular protease, basically, does not require any regulation immediately after its excretia of cells, intracellular proteases are often subjected to more subtle control and regulation.

As for function-specific protease, its localization is often very important, for example, a large number of vacuole-type and periplasmatic proteases involved in the degradation of the protein, whereas membrane-associated proteases play an important role in protein processing (Suarez Rendueles & Wolf, 1988).

A full review of the biological properties and evolution of proteases was published in the work of van den Homberg: Thesis Landbouwuniversitet Wageningen: An analysis of proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, which is introduced in the present description by reference.

Problems associated with the use of proteases

With regard to the application of S. cerevisiae, back in the eighties there were problems and difficulties associated with the use of some proteases, and therefore to solve this problem was developed by a method comprising disruptio target gene target. During secretion, the protein undergoes several proteolytic activity, acting on this secretory pathway. In addition, procuring microorganism, such as Aspergillus, secreted protein can undergo several extracellular proteolytic activities.

Degradation heterological expressed proteins in Aspergillus well covered in the literature (van den Hombergh: Thesis Landbouwuniversitet Wageningen: An analysis of the proteolitic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2), and it was reported that such degradation was observed when the expression of bovine prochimia, the forehead is ekeskog interferon α -2-tHA, GMCSF, IL6, lactoferrin, lysozyme hen egg whites, pork RA, pectin-LiAZ In A. niger, enterotoxin In and β-glucuronidase from E. coli and pectin-LiAZ 3 Erwinia carobotovora.

The problem of proteolysis can be associated with multiple stages of production of the protein. Experts on biological processes that are faced with the problem of proteolysis, can be processed at low temperatures, make early selection of product and protease (protease) or use protease inhibitors. All this can lead to a successful solution of this problem. However, all these difficulties can not be completely eliminated, because, in most cases, degradation occurs in vivo during the production of the protein.

For the implementation of the regulation of proteolysis in the cell, it is important to understand the mechanism of the launch of proteolysis. To a certain extent (heterologous) proteins are proteoliticeski sensitive and recognized as aberrant proteins because of their faulty installation or, in the case of correct installation, as ' foreign ' proteins due to the fact that they do not possess important for their stability characteristics that are specific to the host. Different types of stress can cause an overall significant increase in proteolysis in the cell. Known factors increase the rate of proteolysis are cellular starvation and RA is personal types of stress (i.e. fever, osmotic stress, toxic substances and expression of some heterologous proteins). For the successful solution of problems related to proteolysis in vivo, it was suggested several ways, which will be discussed below. However, the authors of the present invention paid attention to the fact that he could not be absolutely "nefrotoksicskih cells", since proteolysis under the action of intracellular proteases is part of many important metabolic and constitutive reactions (reactions "household"). Therefore, the decrease of the degree of proteolysis is always a process in which there is a change in genetic background, resulting in reduced proteolytic activity, and this process should be analyzed for possible side effects, which can lead to decreased production of the protein (for example, reduction in the rate of growth or sporulation).

Disruptive proteases expressed in filamentous fungi host

Berka and colleagues (1990) described the cloning and disruptive Bottlers Pepa gene .awamori. Recently described three disruptively aspirinplease in A.niger. Were described disruptant for major extracellular aspartate and for the core using aspartate. Double and triple disruptant were generated by recombination and protectyou the s on proteiny range, and on the expression and secretion of pectinase PELB protein A.niger, which is very sensitive to proteolytic degradation (van den Hombergh et al., 1995). Disrupted genes Bottlers Pepa and rerv resulted in reduced activities of these extracellular proteases by 80% and 6%, respectively. In disruptance Δrare, activity other (using) proteases were also significantly reduced, which is caused by inactivation of the proteolytic cascade reactions for other vacuole-type proteases. Reduced extracellular activity correlated with reduced in vitro degradation of PELB and increase in vivo the expression of pelB (van den Hombergh et al., 1996f).

Deficient in protease (prt) mutants of filamentous fungi

Several deficient in protease mutants of Aspergillus have been investigated to increase the level of protein production. Archer and staff was described low level of proteolysis of lysozyme hen egg protein in supernatant prt-double mutant A.niger generated Mattern and co-workers (1992), and it was concluded that although degradation has occurred, but it was significantly reduced. Van den Hombergh et al. (1995) showed that in vitro-degradation of PELB A.niger was reduced in all their seven groups with prt-complementation. In fact prtB-, prtF - prtG-mutants was not observed any degradation. Recently, it was shown that the expression of pelB gene was amplified from six FR is adapted complementarian groups (prtA-F), and the highest expression level was observed in prtB-, prtF - prtG-mutants. In addition to mutants, deficient in one gene, which contained residual extracellular proteolytic activity, ranging from 2% to 80% compared with the activity of wild-type, were generated double mutants through recombination and additional rounds of mutagenesis. These methods were selected and characterized several double prt-mutants, and it was shown that they had found an even lower degree of degradation of the PELB compared with the parent strains.

In addition to suppression by activity by disruptive or mutagenesis, reduced proteolysis can be achieved through negative regulation unnecessary proteolytic activity. This can be accomplished by genetic modification of a promoter or other regulatory sequences of the gene. As shown Fraissinet-Tachet and co-workers (1996), all of extracellular protease in A.niger was regulated by inhibition of carbon catabolite and inhibition of nitrogen metabolite. Cellular starvation also resulted in an overall significant increase in the level of proteolysis in the cell, which was important for cells that do not contain nutrients, but contain proteins that are not necessary under conditions of starvation or are n is necessary only in small quantities. It was reported that when using the strategies of expression, which give high levels of expression in media containing high concentrations of glucose and ammonium, proteolysis decreased. Some constitutive glycolytic promoters (gpd and pkiA) provide a high level of expression in these conditions and can also be used for regulating the expression of (heterologous) gene during continuous fermentation. Type of forced cell starvation can, to varying degrees, influence of various proteases, which indicates the importance of the nutritional status of the cells in this process depending on the type of proteolysis. Therefore, specific proteolysis may be induced by conditions of substrate limitation, which are often used in the processes of large-scale industrial fermentation.

Currently, to solve problems associated with protease, address one or more of the above strategies. However, the residual proteolytic activity yet unidentified proteolytic enzymes is the main problem for professionals. To further reduce the level of unwanted proteolysis, it is imperative to identify new protease responsible for the degradation of homologous and heterologic expressed proteins. The present invention relates to so is m new gene sequences of the protease, coding new protease. As soon as it becomes known primary sequence of a gene of a new protease, one or more of the above strategies recombinant DNA can be used for producing (Ko)mutants with reduced proteolytic activity.

Despite the widespread use of proteases in most industrial processes, known enzymes also have a number of disadvantages relating to at least one of the following properties.

When added to animal feed commonly used protease does not have sufficient resistance to digestive enzymes present in the gastro-intestinal (LCD) tract, for example, pigs or poultry.

With regard to other aspects, the available enzymes do not have sufficient resistance to high temperatures and high pressures that are typically used during operations eviction or deposition.

In addition, commonly used enzymes are not active at the pH range 3-7, i.e. under the conditions of preparation of many foods and beverages, as well as in the LCD tract of most animals.

In accordance with another aspect of the specificity of the commonly used proteases are very limited, which leads to the inability of the present enzymes to break down or dissolve some "proteaseresistant" proteins, and thus to the low yield of the peptides or amino acids. In addition, proteases with low specificity may contribute to the synthesis of new peptides.

Another drawback of the commonly used enzymes is their low specific activity.

Thus, it is obvious that in most cases requires the use of proteases that are more resistant to the digestive enzymes to high temperatures and/or high pressure, and which have new specificnosti in relation to the site of hydrolysis. The present invention relates to such enzymes.

The purpose of the invention

The aim of the present invention to provide a new polynucleotide coding for new protease. Another objective of the present invention to provide a natural and recombinante produced proteases, as well as recombinant strains producing these proteases. Such strains can also be used for producing products in the process of classical fermentation with higher speed or with a higher yield. Another objective of the present invention to provide a strain of filamentous fungi, deficient in the production of the proteases of the present invention. Such strains can be used for more efficient production of heterologous or homologous proteins. On toadie the invention also includes antibodies and hybrid proteins, as well as the methods of obtaining and using polynucleotides and polypeptides of the present invention.

Brief description of the invention

The present invention relates to new polynucleotides coding for new proteases.

More specifically, the present invention relates to polynucleotides having a nucleotide sequence that hybridizes (preferably under conditions of high stringency) with a sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114. In addition, the present invention relates to nucleic acids that are approximately 60%, preferably 65%, more preferably 70%, and even more preferably at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence corresponding to a sequence selected from the group consisting of sequences SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114.

In a more preferred embodiment, the present invention relates to polynucleotide isolated from filamentous fungi, preferably from Aspergillus, in particular, from A.niger.

In one of the embodiments the present invention relates to selected polynucleotide containing the sequence of the nucleic acid, encoding a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171 or its functional equivalent.

In another preferred embodiment, the present invention relates to selected polynucleotide, codereuse at least one functional domain of a polypeptide corresponding to a sequence selected from the group consisting of sequences SEQ ID NO:115 and SEQ ID NO:171 or their functional equivalents.

In a preferred embodiment, the present invention relates to protease gene having a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57. In another aspect the present invention relates to polynucleotide, preferably, to a cDNA that encodes a protease A.niger selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, or to variants or fragments of this polypeptide. In a preferred embodiment of the invention indicated cDNA has a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114 or their functional equivalents.

Genomic clone encoding the polypeptide of the present invention can also be obtained by selection of suitable probes for specific amplification of the genomic region corresponding to any of the sequences SEQ ID NO:1 - SEQ ID NO:5 or its fragments; hybridization of this probe in suitable conditions with genomic DNA obtained from the appropriate body, such as Aspergillus, for example, A.niger, amplification of the desired fragment, for example, by using PCR (polymerase chain reaction), followed by purification and cloning of the amplified fragment.

In an even more preferred embodiment, the present invention relates to polynucleotide comprising the coding sequence of the genomic polynucleotides of the present invention, it is preferred polynucleotide sequence selected from the group consisting of sequences SEQ ID NO:58 - SEQ ID NO:114.

In another preferred embodiment, the present invention relates to cDNA, obtained by cloning and expression of a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57, in a suitable organism, the host, such as A.niger.

The polypeptide of the present invention can also be obtained by cloning and expression of a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 in a suitable organism, the host, such as A.niger.

The present invention also relates to vectors containing the polynucleotide sequence of the present invention and to primers, probes and fragments, which can be used on the I-amplification or detection of the DNA of the present invention.

In another preferred embodiment, the present invention relates to a vector in which the polynucleotide sequence of the present invention is functionally linked to regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable cell host, such as A.niger or A.oryzea. The present invention also relates to methods of obtaining polynucleotides and vectors of the present invention.

In addition, the present invention relates to recombinante produced cells-owners, that contain a heterologous or homologous polynucleotide of the present invention.

In one variation of its implementation of the present invention relates to a recombinant cell host, where significantly reduced expression of the protease of the present invention or reduced its activity, or where this protease is inactivated even. Such recombinants are particularly preferred for the expression of homologous or heterologous proteins.

In another embodiment, the present invention relates to a recombinant cell host, in which the level of expression of the protease of the present invention is significantly increased or increased its activity. Such recombinants are especially predpochtitel is passed for the expression of homologous or heterologous proteins, maturation which is much more difficult when the desired proteolytic cleavage is the rate-limiting stage.

In another embodiment, the present invention relates to recombinante produced the cell host, which contains a heterologous or homologous DNA of the present invention, and preferably, DNA encoding proteins bearing signal sequence, where this cell is capable of producing a functional protease of the present invention, and preferably, to sverkhekspressiya protease of the present invention, where the specified cell is, for example, a strain of Aspergillus, containing an increased number of copies of the gene or cDNA of the present invention.

In another embodiment, the present invention relates to recombinante produced the cell host, which contains a heterologous or homologous DNA of the present invention, where the specified cell can secrete functional protease of the present invention, and preferably, to sverkhekspressiya and to secrete the protease of the present invention, and where the specified cell is, for example, a strain of Aspergillus, containing an increased number of copies of the gene or cDNA of the present invention.

In another aspect the present invention relates to a purified poly is eptide. The polypeptides of the present invention are polypeptides encoded by polynucleotides of the present invention. Especially preferred is a polypeptide having a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171 or its functional equivalents.

The present invention also relates to antibodies that interact with a polypeptide of the present invention. These antibodies can be polyclonal, preferably monoclonal. Such antibodies are particularly suitable for the purification of polypeptides of the present invention.

In the scope of the present invention also includes a hybrid protein containing the polypeptide of the present invention. The present invention also relates to methods of producing the polypeptides of the present invention.

In addition, the present invention relates to a method for the diagnosis of aspergillosis by detection of the presence of the polypeptide of the present invention, or its functional equivalents, or by detection of cDNA of the present invention or fragments or functional equivalents.

The present invention also relates to the use of the protease of the present invention described in this industrial method.

Detailed description of the invention

Polynucleotide

The present invention relates to polynuclear is the Chida, encoding a protease having amino acid sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, or their functional equivalents. The sequence of these genes determined by sequencing genomic clone obtained from Aspergillus niger. The present invention relates to polynucleotide sequences containing the gene encoding these proteases and their complete cDNA sequence and the coding sequence. Accordingly, the present invention relates to selected polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114 or their functional equivalents.

More specifically, the present invention relates to selected polynucleotide, hybridization in harsh environments with polynucleotide selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, preferably, in conditions of high stringency. Preferably, such polynucleotide can be obtained from filamentous fungi, in particular Aspergillus niger. More specifically, the present invention relates to selected polynucleotide having a nucleotide sequence selected from the GRU is dust, consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114.

The present invention also relates to the selected polynucleotide, codereuse at least one functional domain of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171 or their functional equivalents.

Used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, and which include an open reading frame encoding a protein, for example, protease A.niger. A gene may include coding sequences, non-coding sequence, introns or regulatory sequences. In addition, the term "gene" can mean an isolated nucleic acid molecule defined in the present description of the invention.

The nucleic acid molecule of the present invention, such as a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, or its functional equivalents, may be isolated by standard methods used in molecular biology, and using the information presented here concerning katou sequence. So, for example, using all or part of the nucleic acid sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a nucleotide sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, as a hybridization probe, nucleic acid molecules of the present invention can be isolated using standard hybridization techniques and cloning (for example, as described in Sambrook, J., Fritsh, E.F., & Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

In addition, the nucleic acid molecule comprising all or part of the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, can be isolated using polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on sequence information contained in a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114.

Nucleic acid of the present invention can be amplified using cDNA, mRNA or alternatively, genomic DNA as template, and using appropriate oligonucleotide the primers according to standard methods of PCR amplification. Amplificatory thus nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence.

Furthermore, oligonucleotides corresponding to the nucleotide sequences of the present invention or gibridizatsii with them, can be obtained by standard methods of synthesis, for example using an automatic DNA synthesizer.

In a preferred embodiment of the invention selected nucleic acid molecule of the present invention includes the nucleotide sequence presented in the sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114. Sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, corresponds to the coding region cDNA protease A.niger. This cDNA comprises sequences encoding the polypeptide protease A.niger having a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171.

In another preferred embodiment of the invention selected nucleic acid molecule of the present invention includes a nucleic acid molecule that is complementary to the nucleotide sequence presented in the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114 or the functional equivalent of these nucleotide sequences.

A nucleic acid molecule complementary to a different nucleotide sequence, is a molecule that is essentially complementary to a different nucleotide sequence so that it can gibridizatsiya with another nucleotide sequence by formation of a stable duplex.

In one aspect the present invention relates to the selected nucleic acid molecules coding for the polypeptide of the present invention, or its functional equivalent, such as a biologically active fragment or domain, as well as nucleic acid molecules suitable for use as hybridization probes to identify nucleic acid molecules encoding the polypeptide of the present invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.

The terms "isolated polynucleotide" or "isolated nucleic acid" means DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5'-end and the other at the 3'end) in the same genome of an organism, from which it comes. Thus, in one of the embodiments of the invention selected nucleic acid includes some or all of the 5'-non-coding (e.g., promoter) sequences, which are directly adjacent to the coding sequence. Therefore, the term includes, for example, a recombinant DNA which is incorporated into a vector that can replicate offline plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, in the form of cDNA or genomic fragment of DNA produced by PCR or by treatment with restrictase)independent of other sequences. This term also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide, basically, not containing cellular material, viral material, or culture medium (when produced by the methods of recombinant DNA), or chemical precursors or other chemicals (when chemically synthesized). In addition, the term "isolated fragment of a nucleic acid" means a fragment of the nucleic acids that are not usually found in the form of a fragment and not found in nature.

Used herein, the terms "polynucleotide" or "nucleic acid molecule" means m is likely DNA (for example, cDNA or genomic DNA), RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably, it is a double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., iesinovich or phosphorotioate nucleotides). Such oligonucleotides can be used, for example, to obtain nucleic acids, which have an altered ability to form base pairs or increased resistance to nucleases.

In another embodiment of the invention the present invention relates to the selected nucleic acid molecule which is antisense to the nucleic acid molecule protease, for example, the coding chain molecules of nucleic acid protease. In the scope of the present invention also includes a complementary chain described here nucleic acid molecules.

Errors sequencing

The information provided here about the sequence should not be construed in a narrow sense as a requirement for mistakenly identified reason. Described here is a specific sequence can be successfully used in the us to highlight the complete gene from the filamentous fungi, in particular, A.niger, which, in turn, can be easily subjected to further sequence analysis, thereby identifying sequencing errors.

Unless specifically indicated, all nucleotide sequences defined here by sequencing DNA molecules were determined using an automated DNA sequencing machine, and all amino acid sequences of polypeptides encoded defined here by the DNA molecules were predicted by translation of a DNA sequence defined above. Therefore, as known in the art, any defined here the nucleotide sequence of any DNA sequence such automated fashion, may contain a certain number of errors. Typically, the nucleotide sequence defined in an automated fashion, at least about 90%, more preferably at least about 95%-99,9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other means, including methods of DNA sequencing manually. As also known in the art, a single insertion or deletion in a specific nucleotide sequence on sravnenie the actual sequence, will result in a shift in the reading frame during translation of the nucleotide sequence, resulting in a predicted amino acid sequence encoded by a specific nucleotide sequence, will be completely different from the actual amino acid sequence encoded by the sequenced DNA molecule, starting from the point of such insertions or deletions.

Every person is able to identify these correctly identified the reason and fix these errors.

Fragments of nucleic acid probes and primers

The nucleic acid molecules of the present invention may include only a portion or fragment of the nucleic acid sequence presented in the sequence selected from the group consisting of SEQ ID nos:1-57, or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, for example, a fragment that can be used as a probe or primer or a fragment encoding a portion of the protein protease. The nucleotide sequence defined in the cloning of the gene of the protease, and allows you to generate cDNA probes and primers designed for identifying and/or cloning other members of the family of proteases and protease homologues of other species. The probe/primer typically comprises essentially cleared oligonucleic is Ted, usually containing a region of nucleotide sequence that hybridizes, preferably under conditions of high hardness of at least about 12 or 15, preferably about 18 or 20, more preferably from about 22 or 25, and most preferably from about 30, 35, 40, 45, 50, 55, 60, 65 or 75 nucleotides, or more closely spaced nucleotides of the nucleotide sequence presented in the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID nos:58-114, or their functional equivalents.

Probes based on the nucleotide sequences of the protease, can be used to detect transcripts or genomic sequences of the protease encoding the same or homologous proteins that occur, for example, from other organisms. In preferred variants of the invention, the specified probe further includes attached group-label, for example, a band-label can be a radioisotope, fluorescent compound, enzyme or cofactor of the enzyme. Such probes can also be used as part of a diagnostic test kit for identifying cells expressing the protein protease.

Identification and homology

Used herein, the terms "homologue is I" or "percent identity" are used interchangeably. For the purposes of the present invention to determine the percent identity of two amino acid sequences or of two nucleic acid sequences was carried out optimal comparison of sequences (for example, in the sequence of the first amino acid sequence or nucleic acid sequence can be introduced gaps for optimum matching with the second amino acid sequence or nucleic acid sequence). Then a comparison was made between amino acid residues or nucleotides at corresponding positions of the amino acids or in the provisions of nucleotides. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position of the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions for the two compared sequences (that is,% identity = number of identical positions/total number of positions (i.e., overlapping positions)×100).

Preferably, these two sequences have the same length.

Every expert knows that to determine the homology between the two after what euteleostomi there are several different computer programs. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment of the invention, the percent identity between two amino acid sequences is determined using the algorithm of Needleman &Wunsch (J. Mol. Biol. (48):444-453 (1970)), which was introduced in the GAP GCG software (available in http://www.gcg.com) using either matrix Blossom 62 or matrix RAM, and using the weight of the gaps" 16, 14, 12, 10, 8, 6 or 4 and weight length 1, 2, 3, 4, 5 or 6. For professionals it is obvious that all of these different options give slightly different results, but the overall percent identity of two sequences is not significantly changed by using different algorithms.

In another embodiment of the invention, the percent identity between two nucleotide sequences is determined using the GAP program, GCG software (available in http://www.gcg.com) using matrix NWSgapdna. and using the weight of the gaps 40, 50, 60, 70, or 80 and weight length 1, 2, 3, 4, 5 or 6. In another embodiment of the invention, the percent identity of two amino acid or nucleotide sequences is determined using the algorithm of E. Meyer & . Miller (CABIOS, 4:11-17 (1989)which has been incorporated into the ALIGN program (version 2.0) (available in http://vega/igh.cnrs.fr/bin/align-guess.cgi), using weight tables of residues RAM, the penalty for a gap-extension 12 and the fine on a "gap-pass" 4.

Nucleic acid sequence and the protein of the present invention can also be used as the requested sequence for search in the available databases, for example, identify other family members or related sequences. Such searches can be performed using the programs NBLAST and XBLAST (version 2.0)of Altschul et al. (1990) J. Mol. Biol. 215:403-10. Search nucleotides in the BLAST can be performed using the NBLAST program, the label for count = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the protease of the present invention. The search for proteins in the BLAST can be carried out using the XBLAST program, the label for count = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules protease of the present invention. To implement the mapping with the introduction of the "gaps"can be used the BLAST program with the entered "porous", as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the programs BLAST and BLAST entered porous, can be IP is alsomany parameters of the respective programs by default (for example, XBLAST and NBLAST). Cm. http://www.ncbi.nim.nih.gov.

Hybridization

Used herein, the term "hybridization" refers to conditions for hybridization and washing under which nucleotide sequences that are at least about 50%, at least about 60%, at least about 70%, more preferably at least 80%, even more preferably at least about 85-90%, and most preferably at least 95% homologous to each other, hybridize with each other.

Preferred non-limiting example of such hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°followed by one or more washes in 1×SSC, 0,1% - ordinator at 50°C, preferably at 55°more preferably, at 60°and even more preferably 65°C.

Conditions of high stringency are, for example, hybridization at 68°5×SSC/5H denhardt's solution/1.0% of LTOs and wash at 0.2×SSC/0,1% - ordinator at room temperature. Alternatively, washing can be carried out with 42°C.

Each specialist is known, in which cases it is necessary to apply strict terms & conditions of high stringency hybridization. For more information about these conditions can be found in the literature, for example, the manual Sambrook et al., 1989, olecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology (John Wiley & Sons, N.Y.).

Needless to say that polynucleotide that hybridizes only with poly(A)sequence (such as 3'-terminal poly(A)tail of mRNA) or a complementary fragment of the residues of T (or U), is not included in polynucleotide of the present invention used for specific hybridization with a part of the nucleic acid of the present invention, since such polynucleotide should gibridizatsiya with any nucleic acid molecule containing a poly(A)tail or a complementary sequence (for example, with any double-stranded DNA clone).

Obtaining full-length DNA from other organisms

Conventional techniques may be skanirovaniya cDNA libraries constructed from other microorganisms, for example, filamentous fungi, and in particular, from the fungi Aspergillus species.

For example, strains of Aspergillus can be skanirovaniya on homologous polynucleotide protease using Northern blot analysis. After detection of transcripts homologous to polynucleotides of the present invention, cDNA libraries can be constructed from RNA extracted from the corresponding strain by standard methods, well known to specialists. Alternatively, the entire genomic DNA library can be skriner the van with the use of a probe, hybridizing with polynucleotides protease of the present invention.

Homologous gene sequences can be selected, for example, by PCR using two oligonucleotide primers or two pools of degenerate oligonucleotide primers designed on the basis described herein nucleotide sequences.

Matrix for the reaction may be cDNA obtained by reverse transcription of mRNA isolated from strains that are known or assumed to Express polynucleotide of the present invention. The PCR product may be subcloned and sequenced to ensure that these amplificatoare sequences are sequences of nucleic acids of a new protease or its functional equivalent.

The PCR fragment can then be used to highlight the full-size cDNA clone various known methods. For example, amplificatory fragment can be isolated and used for screening bacteriophage or kosmidou cDNA library. Alternatively, the labeled fragment can be used for screening of the genomic library.

For selection of full-length cDNA sequences from other microorganisms can be used PCR technology. For example, RNA can be isolated in soo is according to standard procedures, from an appropriate cellular or tissue source. To start the synthesis of the first chain reaction reverse transcription can be performed on the RNA using oligonucleotide primers that are specific mainly to the 5'-end of the amplified fragment.

The resulting hybrid DNA/RNA can then be "lengthened" (e.g., guanine) via a standard terminal transferase reaction, and this hybrid can be cleaved by RNase H, after which it can be started a second synthesis circuit (for example, using a poly-C primer). Thus, cDNA sequences, located above the amplified fragment can be easily selected. An overview of suitable cloning strategies can be found, for example, Sambrook et al., see above; and Ausubel et al., see above.

Vectors

In another aspect the present invention relates to vectors, preferably by expressing vectors, containing a nucleic acid encoding a protein, protease or its functional equivalent. Used herein, the term "vector" means a nucleic acid molecule that can carry another nucleic acid to which it was attached. One type of vector is a "plasmid", which is a circular double-stranded DNA loop into which can be legirovanyh additional DNA segments. Another type of vector is a viral vector, where the incremental DNA segments can be legirovanyh in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are introduced (e.g., bacterial vectors having a bacterial replication source, and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) are integrated into the genome of a host cell upon introduction into the cell, and thereby are replicated along with the genome of the host. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to here as "expressing vectors". Basically, expressing vectors are often used in the technique of recombinant DNA in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably as the plasmid is the most common form of the vector. However, the present invention applies to other forms expressing vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which have equivalent functions.

Recombinant expressing the vectors of the present invention include nucleic acid of the present invention in a form suitable for expression of this nucleic acid in the cell host, which means that expressing recombinant vector including the et one or more regulatory sequences, selected in accordance with the used for the expression of cell-owners and functionally related to expressed sequence nucleic acids. Used in relation to expressing recombinant vector, the term "functionally connected" means present therein and interest nucleotide sequence attached to the regulatory sequence(s) in a manner that ensures the expression of this nucleotide sequence (e.g., in an in vitro system of transcription/translation or in the cell-the owner, if the specified vector is introduced into this cell the owner). The term "regulatory sequence" refers to the promoters, enhancers and other regulatory expression elements (e.g., polyadenylation signal). Such regulatory elements are described, for example, Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences are sequences which direct constitutive expression of a nucleotide sequence in the cells are the owners of many types, and sequences that direct expression of the nucleotide sequence only in certain cells of the host (for example, tissue-specific regulatory sequences). However, it should be noted that the design of expressing what about the vector may depend on such factors as the selection of transformed host cells, the level of expression of the desired protein, etc. Expressing the vectors of the present invention can be introduced into cells of a host, as a result they will produce proteins or peptides, encoded described here nucleic acids (e.g., proteins, proteases, mutant forms of proteins proteases, their fragments, variants or functional equivalents, hybrid proteins and the like).

Recombinant expressing the vectors of the present invention can be designed for expression of proteins of proteases in prokaryotic or eukaryotic cells. So, for example, proteins, proteases can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expressing vectors), yeast cells or mammalian cells. Appropriate cell hosts are discussed in detail in the work Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, recombinant expressing the vector can be transcribed and translated in vitro using a promoter regulatory sequence T7 and T7 polymerase.

Expressing the vectors used in the present invention, are chromosomal, epilimnia and viral vectors, e.g., vectors derived from bacterial plasmids, the tank is of eritage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papovavirus, vaccinia viruses, adenoviruses, poxviruses poultry, pseudorapidity and retroviruses, and vectors derived from combinations thereof, such as vectors derived from plasmids and bacteriophobic genetic elements, such as Comedy and family.

The DNA insert should be functionally attached to a suitable promoter, such as the PL promoters of phage lambda, the lac promoters, trp and tac E. coli, the early and late SV40 promoters and promoters of retroviral LTRS, etc. well known to Experts and other suitable promoters. In a specific embodiment of the invention preferred are promoters capable of high level expression of proteases in filamentous fungi. Such promoters are known in the art. Expressing constructs may contain sites of transcription initiation, termination of transcription, and transcribed region of the binding site to the ribosome for translation. The coding portion of the Mature transcripts expressed by these structures includes the site of translation initiation AUG at the beginning of the sequence and the codon translation termination, located in a suitable position at the end of the translated polypeptide.

Vector DNA can be introduced into prokaryotic the ski or eukaryotic cells by standard methods of transformation or transfection. Used herein, the terms "transformation" and "transfection" means the number of well-known specialists of methods for introducing foreign nucleic acid (e.g., DNA) into the cell host, including co-precipitation with calcium phosphate or calcium chloride mediated by DEAE-dextran transfection, transduction, infection, lipofection mediated by cationic lipid transfection or electroporation. Suitable methods of transformation or transfection of host cells can be found in the manual in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986)and other laboratory manuals.

As for stable transfection of mammalian cells, it is known that only a small fraction of cells may integrate the foreign DNA into their genome, depending on expressing vector and transfection method. For identification and selection of these integrants, the gene encoding a selective marker (e.g., a marker of resistance to antibiotics), usually injected into the cells of the hosts along with the desired gene. Preferred selective markers are markers that give resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selective marker can be introduced into the cell-Ho is lain on the same vector, which encodes a protein protease, or it can be introduced on a separate vector. Cells stably transfetsirovannyh introduced nucleic acid can be identified by selection for resistance to a drug (e.g., cells, which was introduced in the selective marker gene, survive, while the other cells die).

The expression of proteins in prokaryotes, in most cases, is carried out in E.coli cells comprising vectors containing constitutive or inducible promoters regulating expression of hybrid or non-hybrid proteins. Hybrid vectors add a number of amino acids to be encoded protein in it, for example, to aminobenzo recombinant protein. Such hybrid vectors typically serve three purposes, namely: 1) enhance the expression of recombinant protein; 2) increase the solubility of the recombinant protein; and 3) facilitate the purification of recombinant protein by acting as a ligand in affinity purification. In most cases, hybrid expressing vectors, the site of proteolytic cleavage is injected at the junction of the hybrid molecule and recombinant protein that allows to separate the recombinant protein from the hybrid molecules, and then spend cleaning this hybrid protein. Such enzymes and their sequences cognatio recognition are factor XA, thrombin, and e is deracinate.

As mentioned above, expressing the vectors preferably contain a selective marker. Such markers are gene digidrofolatreduktazy or gene of resistance to neomycin for eukaryotic cell culture, and the gene of resistance to tetracycline or ampicillin for culturing in E. coli and other bacteria. Representative examples of suitable host is a bacterial cell such as E. coli, Streptomyces and Salmonella typhimurium; cells of fungi, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as Cho, COS and melanoma Bowes; and plant cells. Suitable culture medium and culturing conditions the above host cells known in the art.

Vectors preferred for use in bacteria are the vector pQE70, pQE60 and PQE-9, supplied from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, supplied from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 supplied from Pharmacia. Preferred eukaryotic vectors are the vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG supplied from Stratagene; and the vector pSVK3, pBPV, pMSG and pSVL supplied from Pharmacia. Other suitable vectors are well known to specialists.

Known bacterial promoters used in the present invention are promoters lacl and lacZ E. coli promoters, the T3 and T7, gpt promoter, the PR, the motors lambda PR, the PL promoter and the trp promoter, the promoter timedancing HSV, early and late SV40 promoters, the promoters LTR of retroviruses such as rous sarcoma virus ("RSV"), and the promoters of metallothionein, such as the promoter of the mouse metallothionein-1.

Transcription of DNA encoding the polypeptides of the present invention, in higher eukaryotes can be increased by introducing a vector of the enhancer sequence. Enhancers are CIS-acting elements of DNA, usually have a length of approximately 10-300 BP and to enhance the transcriptional activity of the promoter in a cell-the owner of this type. Examples of enhancers are the SV40 enhancer, located in the later section of the site of replication initiation and having a length BP 100-270, early enhancer promoter and cytomegalovirus enhancer of polyoma located in the later section of the site of replication initiation, and adenovirus enhancers.

For secretion of the translated protein into the area of the lumen of the endoplasmic reticulum, periplasmatic space or in the extracellular space, expressed polypeptide can be introduced corresponding signal secretion. These signals may be endogenous to the polypeptide, or they may be heterologous.

The polypeptide can be expressed in a modified form, such as a hybrid protein, and may in luceti not only secretion signals, but also additional heterologous functional regions. For example, the area consisting of other amino acids, particularly charged amino acids, may be added to the N-end of the polypeptide to improve its stability and sustainability in the cell-master in the cleaning process or during subsequent processing and storage. In addition, peptide molecules can be added to the polypeptide to facilitate purification.

The polypeptides of the present invention

The present invention relates to the selected polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171; amino acid sequence, obtained by expression of polynucleotide of the present invention, or, in a preferred embodiment of the invention, a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 in the respective owner, as well as amino acid sequence, obtained by expression of a polynucleotide sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114 in the corresponding master. In addition, the scope of the present invention includes a peptide or polypeptide containing the functional equivalent of the above polypeptides. The above polypeptides, in General, are referred to as "polypeptides of the present invention".

ermine "peptide" and "Oligopeptide" are treated as synonyms (in the conventional sense), and these terms are used interchangeably depending on the context where you want to specify that the chain consists of at least two amino acids linked patibility links. Used herein, the term "polypeptide" means a circuit that contains more than seven amino acid residues. All Oligopeptide and polypeptide of the formula or their sequences are written left to right, in the direction from aminobenzo to carboxilic. Single-letter code is used amino acids are well known in the art and can be found in the manual, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

The term "isolated" polypeptide or protein is meant a polypeptide or protein extracted from its natural environment. For example, recombinante produced polypeptides and proteins expressed in the cells of the host, are allocated for the purposes of the present invention, as it was defined for native or recombinant polypeptides which have been essentially purified by any appropriate means, such as, for example, the method of one-step purification, described Smith & Johnson, Gene 67:31-40 (1988).

The protease of the present invention can be isolated and purified from recombinant cultures of well-known methods, including precipitation with ammonium sulfate or ethanol, ek is the traction acid, anyone - or cation-exchange chromatography, chromatography on phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, chromatography on hydroxyappatite and chromatography on the lectin. For purification for analytical purposes, it is most preferable to use high performance liquid chromatography ("HPLC").

The polypeptides of the present invention are the naturally purified products, products of chemical synthesis and products produced by recombinant techniques from a prokaryotic or eukaryotics owners, including, for example, bacterial cells, yeast cells, plant cells, insect cells and mammalian cells. Depending on the host in the methods of recombinant production of polypeptides of the present invention can be glycosylated or deglycosylation. In addition, the polypeptides of the present invention may also include modified initiating meinenemy the remainder, which, in some cases, results from a cellular-mediated processes in the host.

In addition, the protein of the present invention may be the precursor protein such as zymogen; hybrid protein; protein obtained in the form of Pro-sequence or pre-Pro-sequence, or other immature forms l is the God type.

The protein fragments

The present invention also relates to biologically active fragments of the polypeptides of the present invention.

Biologically active fragments of the polypeptides of the present invention are polypeptides comprising the amino acid sequence that is essentially identical to the amino acid sequence of the protein protease or come from this sequence (for example, amino acid sequence contained in a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171) and which include fewer amino acids than the full-size protein, and possess at least one biological activity corresponding to the activity of the full-size protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the protein protease. Biologically active fragment of the protein of the present invention may be a polypeptide, which has a length of, for example, 10, 25, 50, 100 or more amino acids. Moreover, other biologically active portion in which deleterows other areas of the protein, can be obtained by recombinant techniques and evaluated for one or more of the biological activities inherent in the native form of the polypeptide of the present invention.

The present invention t is the train refers to fragments of the nucleic acid, which encode the above-described biologically active fragments of the protein protease.

Hybrid proteins

Proteins of the present invention or their functional equivalents, for example, a biologically active portion may be functionally attached to nepotismo the polypeptide (e.g., heterologous amino acid sequences) to produce hybrid proteins. Used herein, the term "chimeric protein" or "hybrid protein" means protease polypeptide protease, functionally attached to nepotismo the polypeptide. The term "proteiny polypeptide" means a polypeptide having the amino acid sequence corresponding to the polypeptide sequence of the present invention, and the term "nepotianus polypeptide" means a polypeptide having the amino acid sequence of the corresponding protein, which essentially is not homologous to the protein of the present invention, for example, a protein that is different from proteasome protein and is derived from the same or another organism. In protease hybrid protein proteiny polypeptide can correspond to the entire protein or part protein of the present invention. In a preferred embodiment of the invention a hybrid protein protease comprises at least one biologically active fragment of a protein N. the present invention. In another preferred variant of the invention, the hybrid protein protease comprises at least two biologically active portion of a protein of the present invention. Used in relation to hybrid protein, the term "functionally connected" indicates that proteiny polypeptide and nepotianus polypeptide fused with each other with preservation of the reading frame. Nepotianus polypeptide can be fused to the N-end or From the end proteasome polypeptide.

For example, in one of the embodiments of the invention, the hybrid protein is a hybrid protein "GST-protease", where these protease sequence fused with the end of the GST sequences.

Such hybrid proteins can facilitate the purification of recombinant protease. In another embodiment of the invention, the hybrid protein is proteiny protein containing a heterologous signal sequence at its N-Terminus. In some cells of the host (for example, in mammalian cells and in yeast cells, host) expression and/or secretion of the protease can be enhanced with the use of a heterologous signal sequence. In another example, a secretory sequence, gp67 protein shell of baculovirus can be used as a heterologous signal sequence (Curren Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences are secretory sequence Meletina and alkaline phosphatases of human placenta (Stratagene; La Jolla, California). Another example of suitable prokaryotic heterologous signal sequences are secretory signal phoA (Sambrook et al., see above) and secretory protein signal (Pharmacia Biotech; Piscataway, New Jersey).

The signal sequence can be used to facilitate secretion and protein or polypeptide of the present invention. Signal sequences are usually characterized by a core of hydrophobic amino acids, which are usually hatshepsuts from the Mature protein during the secretion of one or more of the events off. Such signal peptides contain processing sites that allow you to split the signal sequence from the Mature proteins as they pass through the secretory pathway. The signal sequence directs the secretion of the protein, for example, from a eukaryotic host, which was introduced expressing vector, and thereafter or simultaneously, the signal sequence is cleaved. Then the protein can be easily purified from the extracellular environment known methods. Alternatively, the signal sequence may shall be attached to the desired protein using sequence which facilitates cleaning, for example, using the GST domain. For example, a sequence encoding a polypeptide may be fused to a marker sequence, for example, with a sequence that encodes a peptide that facilitates purification of the hybrid polypeptide. In some preferred embodiments of this aspect of the present invention, the marker sequence is exegetically peptide, such as a label, is present in the vector pQE (Qiagen, Inc.), other labels, many of which are commercially available. As, for example, described in Gentz et al., Proc.Natl. Acad. Sci., USA 86:821-824 (1989), hexastylis provides for convenient purification of the hybrid protein. The label ON is another peptide that is used for cleaning, which corresponds to the epitope, derived from protein hemaglutinin influenza virus, which has been described, for example, Wilson et al., Cell 37:767 (1984).

Preferably, the chimeric or hybrid protein protease of the present invention is obtained using standard techniques of recombinant DNA. For example, DNA fragments coding for different polypeptide sequences are ligated together in the same frame is read by standard methods, for example, using a blunt or sticky ends for ligation, then break down restrictase obtaining from the respective ends, and if necessary, complete sticky ends, treated with alkaline phosphatase in order to avoid undesirable joining, and subjected to enzymatic legirovanie. In another embodiment of the invention, the hybrid gene can be synthesized by standard methods, including methods using automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers are designed so that they are complementary protruding ends, between two adjacent gene fragments, which can then be hybridized and re-amplified with obtaining a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons, 1992). In addition, many expressing vectors are commercially available and encode hybrid part (e.g., a GST polypeptide). Encoding the protease nucleic acid can be cloned into the specified expressing vector so that this hybrid part was annexed to proteasome squirrel with preservation of the reading frame.

Functional equivalents

The terms "functional equivalents" and "functional variants" are interchangeable. Functional equivalents of the DNA of the present invention are selected Incrament, encoding a polypeptide, which has a specific function defined here protease A.niger. The functional equivalent of the polypeptide of the present invention is a polypeptide that has at least one function defined here protease A.niger.

Functional equivalents of the protein or polypeptide can contain only conservative substitution of one or more amino acids in a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, or substitutions, insertions or deletions of essential amino acids. In line with this, nonessential amino acid residue is a residue that can be replaced in a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, without significant changes in biological function. For example, it is assumed that especially unsuitable for modification are conservative amino acid residues protease proteins of the present invention. Alternatively, conservative amino acids in protease proteins of the present invention and other proteases are also unsuitable for modification.

The term "conservative substitution" means the replacement, in which the amino acid residue substituted amino acid residue having a similar side chain. These families of amino acids known in the art and include amino is islote with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Functional equivalents of the nucleic acid may mainly contain silent mutations or mutations that do not alter the biological function of the encoded polypeptide. In accordance with this present invention relates to nucleic acid molecules coding for protease proteins that contain amino acid residues, largely affect specific biological activity. Such protease proteins by their amino acid sequence different from a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, however, they retain at least one biological activity. In one of the embodiments of the invention selected nucleic acid molecule includes a nucleotide sequence encoding a protein, where this protein with the holding amino acid sequence, essentially homologous, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequences presented in the sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171.

So, for example, guidance on the implementation of phenotypically silent amino acid substitutions can be found in the work of Bowie J.U. et al., Science, 247:1306-1310 (1990), where the authors indicate that there are two main methods of examination of tolerance amino acid sequence substitutions. The first method is based on the process of evolution, in which mutations or acquired, or suppressed as a result of natural selection. The second method involves genetic engineering to introduce amino acid substitutions at specific positions of a cloned gene, and selection or screening for the identification of sequences that retain their functional properties. According to the authors, these studies unexpectedly revealed that proteins are tolerant to amino acid substitutions. In addition, the authors indicate that these changes are likely to be valid in a certain position of a given protein. For example, the most hidden of amino acid residues required nonpolar side chains, whereas some elements of the surface side chains are mainly konservat the ate. Other such phenotypically silent substitutions described, for example, Bowie et al., see above, and listed in the references.

The selected nucleic acid molecule encoding proteiny protein homologous to a protein selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, can be constructed by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, so that one or more amino acid substitutions, deletions or insertions were introduced into the encoded protein. These mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

The term "functional equivalents" also covers orthologues proteasome protein A.niger. Orthologues proteasome protein A.niger represent proteins that can be isolated from other strains or species, and which have a similar or identical biological activity. Such orthologues can be easily identified as containing amino acid sequence essentially homologous to a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171.

Used herein, the term "essentially homologous" about what is worn to the first amino acid or nucleotide sequence, which contains a sufficient or minimum number of amino acids or nucleotides that are identical or equivalent (e.g., with a similar side chain) amino acids or nucleotides second amino acid or nucleotide sequence, so that these first and second amino acid or nucleotide sequences have a common domain. For example, amino acid or nucleotide sequences that contain a common domain, identical to about 60%, preferably 65%, more preferably 70%, and even more preferably at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and are as defined above, quite identical.

In addition, the scope of the present invention includes nucleic acids encoding other members proteases family, which have a nucleotide sequence differing from the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114. In addition, the scope of the present invention includes nucleic acids encoding protease proteins from other species, which have a nucleotide sequence differing from the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114.

The Molek is s nucleic acid, appropriate options (for example, natural allelic variants and homologues of the DNA protease of the present invention, can be isolated based on their homology with respect to the described here nucleic acid protease, using cDNA described here or suitable fragments, as a hybridization probe according to standard hybridization methods, preferably, hybridization conditions of high stringency.

Experts know that in addition to the use of natural allelic variants of the sequence of the protease, the nucleotide sequence contained in a sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, may be made of modifications, which lead to changes in the amino acid sequences of the protease, but not noticeably affect the function proteasome protein.

In another aspect the present invention relates to improved protease proteins. "Improved" protease proteins are proteins that have at least one improved biological activity. Such proteins can be obtained by introducing non-specific mutations throughout the coding sequence of the protease or its part, for example, what Ecodom saturating mutagenesis, and the resulting mutants can be recombinante expressed and skanirovaniya on their biological activity. For example, the literature describes the standard tests for measuring the enzymatic activity of proteases and improved proteins can be easily selected.

In a preferred embodiment of the invention proteiny protein has an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171. In another embodiment of the invention the polypeptide protease, mostly homologous amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, and retains at least one biological activity of a polypeptide corresponding to a sequence selected from the group consisting of SEQ ID NO:115-171, but it differs in its amino acid sequence due to natural changes or mutagenesis, as described above.

In yet another preferred embodiment of the invention proteiny protein has the amino acid sequence encoded by the selected nucleic acid capable of gibridizatsiya with nucleic acid corresponding to a sequence selected from the group consisting of the C SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58 - SEQ ID NO:114, preferably, hybridization conditions of high stringency.

In accordance with this proteiny protein is a protein comprising amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence presented in the sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, and retains at least one functional activity of the polypeptide corresponding to the sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171.

Functional equivalents of the protein of the present invention can also be identified, for example, by screening combinatorial libraries of mutants, e.g., a truncated mutant protein of the present invention, protease activity. In one of the embodiments of the invention generiert discrete library of variants by combinatorial mutagenesis at the level of nucleic acids. Such a discrete library of variants can be produced, for example, by enzymatic ligating a mixture of synthetic oligonucleotides into gene sequence, so that the degenerate series of potential protein sequences of expressi who was ovalis as individual polypeptides, or alternatively, as a set of larger hybrid proteins (e.g. for phage view). To produce libraries of potential variants of the polypeptides of the present invention from a degenerate oligonucleotide sequence can be used a number of methods. Methods for the synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of the polypeptide of the present invention can be used to generate a discrete population of polypeptides for screening and subsequent selection of variants. So, for example, a library of fragments of the coding sequence can be generated by treating the double-stranded PCR fragment of a desired coding sequence of the nuclease under conditions in which one molecule has only one single-strand break ("nick"); by denaturing the double-stranded DNA; renaturation of DNA with the formation of double-stranded DNA which can include sense/antisense pairs from different nick-products; removal of single-stranded fragments of the newly formed duplexes by treatment with S1 nuclease, and ligating the resulting library of fragments in expressyou the s vector. This method can be obtained expression library that encodes N-terminal and internal fragments of the desired protein in various sizes.

Specialists are known several methods for screening gene products of combinatorial libraries, obtained by point mutations with truncation, and screening cDNA libraries for gene products having a selected property. Methods suitable for high-performance analysis and the most widely used for screening large gene libraries typically include cloning the gene library into replicable expressing vectors, transforming appropriate cells received by the library of vectors, and the combined expression of the genes under conditions in which detection of a desired activity facilitates allocation vector containing the gene, the products of which were detected. Recursive multiple mutagenesis (REM), which is a way of increasing the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of the protein of the present invention (Arkin & Yourvan (1992) Proc.Natl. Acad. Sci., USA, 89:7811-7815; Delgrave et al. (1993), Protein Engineering 6(3):327-331).

In addition to the sequence proteasome gene sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO:57, for special is the aliste obviously, in this population may be a polymorphism of DNA sequence, which can lead to changes in the amino acid sequence proteasome protein. Such genetic polymorphism may be present in cells of different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.

Fragments of polynucleotide of the present invention may also include polynucleotide that do not encode functional polypeptides. Such polynucleotide can function as probes or primers for the PCR reaction. Such polynucleotide can also be used if it is desirable to prevent the functional activity of the protease in a particular organism ("knockout"mutants).

Nucleic acids of the present invention, regardless of whether they encode a functional or non-functional polypeptides, can be used as hybridization probes or primers in polymerase chain reaction (PCR). The use of the nucleic acid molecules of the present invention that do not encode a polypeptide having by activity includes, among other things : (1) isolation of the gene encoding proteiny protein, or its allelic variants of the cDNA library, the example other organisms that are not A.niger; (2) in situ hybridization (eg, FISH) with preparations of metaphase chromosomes in order to provide precise chromosomal localization of the gene of the protease, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting mRNA expression of the protease in specific tissues and/or cells, and (4) the possible use of probes and primers as a diagnostic tool for analyzing for the presence of nucleic acids, hybridizing with protease probe in a given biological sample (e.g. tissue).

In the scope of the present invention also includes a method of obtaining a functional equivalent of a protease gene or cDNA. This method involves obtaining a labeled probe, which includes the selected nucleic acid encoding all or part of the sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171 or her option; screening the library of nucleic acid fragments using a labeled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library with the formation of nucleic acid duplexes; and producing the full gene sequence from the nucleic acid fragments in any labeled duplex to obtain a gene, umstvennogo protease gene.

In one embodiment of the invention nucleic acid protease of the present invention, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous nucleic acid sequence presented in the sequence selected from the group consisting of SEQ ID NO:1 - SEQ ID NO:57 or a sequence selected from the group consisting of SEQ ID NO:58-SEQ ID NO:114 or them complementary sequences.

In another preferred embodiment, the polypeptide protease, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence presented in the sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171.

Cell-hosts

In another embodiment, its implementation of the present invention relates to cells, for example, transformed cells-owners or to the recombinant cell host, which contain the nucleic acid included in the scope of the present invention. The term "transformed cell" or "recombinant cell" means a cell (or its predecessor), which, by the methods of recombinant DNA introduced nucleic acid of the present invention. Prokaryotic and eukaryotic cells are, for example, bacteria, fungi, yeast and the like, and particularly preferred cell and are cells of filamentous fungi, in particular, Aspergillus niger.

A host cell may be selected so that it was carried out modulation of the expression of the built-in sequences, or carried out the modification and processing of the gene product in accordance with specific desired mechanism. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate the optimal functioning of the protein.

Different cell owners have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or system owners, well-known specialists in the field of molecular biology and/or Microbiology, may be selected so that they provide the necessary and correct modification and processing of the expressed foreign protein. For this purpose, can be used eukaryotic cell hosts with cellular mechanism for ensuring that the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product. These cells are the owners of well known professionals.

Cells also hosts include, but are not limited to, mammalian cell lines such as Cho, VERO, KSS, HeLa, COS, MDCK, 293, T, and WI38 cell lines harodnogo plexus.

If the need is about, the polypeptides of the present invention can be produced by using stably transfected cell lines. There are a number of vectors suitable for stable transfection of mammalian cells and is accessible to specialists and well-known methods for constructing such cell lines are described, for example, Ausubel et al. (see above).

Antibodies

In addition, the present invention relates to antibodies, such as monoclonal or polyclonal antibodies that specifically bind to proteins protease of the present invention.

Used herein, the terms "antibody" (Ab) or "monoclonal antibody" (mAb) mean intact molecules as well as antibody fragments (such as Fab and F(ab')2-fragments), which can specifically bind with protease protein. Fab and F(ab')2fragments do not contain the Fc fragment of intact antibody; quickly removed from the bloodstream and can have less nonspecific binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). And so the fragments are preferred.

Antibodies of the present invention can be obtained by any means. For example, cells expressing proteiny protein or antigenic fragment can be introduced animal to stimulate the production of sera containing polyclonal the specific antibodies. In a preferred method get and purify the drug proteasome protein, which essentially does not contain natural impurities. Then this drug is administered to an animal for the production of polyclonal antisera with a higher specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or fragments thereof that communicates with protease protein). Such monoclonal antibodies can be produced using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). Basically, such procedures involve immunizing an animal (preferably a mouse) antigen proteasome protein, or a cell expressing proteiny protein. Splenocytes of these mice is isolated and subjected to fusion with a suitable myeloma cell line. In accordance with the present invention can be used with any suitable myeloma cell line, however, it is preferable to use the parent myeloma cell line (SP2O), available at the American type culture collection, Rockville, PCs Maryland. After merging the obtained hybridoma cells survive in the selective medium NAT, and then clone by limited dilution as described by Wands et al. (Gastr-enterology, 80:225-232 (1981)). Hybridoma cells obtained through such a selection are then analyzed to identify clones that secrete an antibody able to bind to the antigen proteasome protein. Generally speaking, these polypeptides can be attached to a protein-carrier, such as KLH, as described in Ausubel et al. (see above), mixed with adjuvant and injected the mammalian host.

In particular, various animal hosts can be immunitary by injection of the desired polypeptide. Examples of suitable host animals include rabbits, mice, Guinea pigs and rats. To increase the immunological response can be used in different adjuvants depending on the type of owner, including, but not limited to, beta-blockers (complete and incomplete), adjuvant based on mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols-pluronic, polyanion, peptides, oil emulsions, hemocyanin lymph snails, dinitrophenol, BCG (Bacillus of Calmet-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules present in the serum of immunized animals.

Such antibodies may be of any immunoglobulin classes IgG, IgM, IgE, IgA, IgD and any of their subclasses. Hybridoma producing mAb of this image is etenia, can be cultivated in vitro or in vivo.

After the production of polyclonal or monoclonal antibodies are tested for specific recognition proteasome polypeptide or its functional equivalent in the immunoassay, such as Western blot analysis or analysis on immunoprecipitation using standard techniques, for example, described in Ausubel et al. (see above). In the present invention can be used antibodies that specifically associated with ProcessName proteins or their functional equivalents. For example, such antibodies can be used in the immunoassay for detection of protease in pathogenic or non-pathogenic strains of Aspergillus (e.g., extracts of Aspergillus).

Antibodies of the present invention is preferably produced using a polypeptide fragments of the protease, which probably are also antigenic, as indicated by criteria such as high frequency of charged residues. For example, such fragments can be generated by standard PCR methods, and then expressing cloned in the vector pGEX (Ausubel et al., see above). Then the hybrid proteins can be expressed in E. coli and purified using affinity matrix with glutathione-agarose as described by Ausubel et al. (see above). If necessary, each protein can be generated by a number of the (for instance, two or three) hybrids, and each hybrid can be injected at least two rabbits. Anticavity can be produced by successive injections, usually at least three booster injections. Basically, anticigarette evaluated on its ability to thus recombinant protease polypeptide or its functional equivalents, whereas the unbound proteins can serve as a control to assess the specificity of the immune response.

Alternatively, techniques described for the production of single-chain antibodies (U.S. patent No. 4946778 and 4704692), can be adapted to produce single-chain antibodies against proteases polypeptide or its functional equivalents. There are commercially available kits for generating and screening phage libraries presentation, delivered, for example, the firm Pharmacia.

In addition, examples of methods and reagents particularly suitable for use in generating and screening libraries of view of antibodies can be found, for example, in U.S. patent No. 5223409; in PCT publication no WO 92/18619; in PCT publication no WO 91/17271; in PCT publication no WO 20791; in PCT publication no WO 92/20791; in PCT publication no WO 92/15679; in PCT publication no WO 93/01288; in PCT publication no WO 92/01047; in PCT publication no WO 92/09690; in PCT publication no WO 90/02809; Fuchs et al. (1991) Bio/Technology, 9:370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas, 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

Polyclonal and monoclonal antibodies that specifically associated with protease polypeptides or their functional equivalents, can be used, for example, for detection of gene expression of the protease or its functional equivalent, for example, in another strain of Aspergillus.

For example, the protease polypeptide can be easily detected in standard immunoassays cells or extracts of Aspergillus. Examples of suitable assays include, but are not limited to, Western blotting, ELISA, radioimmunoassay etc.

The term "specific binding" means that the antibody recognizes a specific antigen and associated with, for example, the protease polypeptide, and, in fact, does not recognize other unrelated molecules in the sample and is not associated with them.

Antibodies can be purified, for example, methods of affinity chromatography in which the polypeptide antigen is immobilized on the resin.

Antibody directed against a polypeptide of the present invention (e.g., monoclonal antibody)can be used to highlight the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. In addition, this antibody can be used for detection of the protein (for example,in a cellular lysate or cell supernatant) to assess the level and nature of the expression of this polypeptide. These antibodies can also be used as a diagnostic tool for monitoring levels of proteins in cells or tissues, as part of a clinical testing procedure, for example, to determine the effectiveness of this scheme of treatment or for diagnosis of aspergillosis.

Detection can be facilitated by binding of the antibody with detektivami substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetyltransferase; examples of suitable fluorescent materials are umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorofluorescein, disilgold or phycoerythrin; an example of a luminescent material is luminal; examples of bioluminescent materials include luciferase, luciferin, and acorin, and suitable examples of radioactive materials are125I131I35S or3N.

Preferred epitopes covered by the antigenic peptide are regions, localized on the surface of the protein, e.g., a hydrophilic region. Graphics hydrophobicity proteins nastoyascheevremya can be used to identify the hydrophobic areas. The antigenic peptide of a protein of the present invention includes at least 7 (preferably 10, 15, 20 or 30 contiguous amino acid residues of the amino acid sequence selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:171, and contains the epitope of the protein, which stimulates the production of antibodies against the peptide, forming a specific immune complex with the protein.

Preferred epitopes covered by the antigenic peptide is a region of the protease that is localized on the surface of the protein, e.g., hydrophilic regions, hydrophobic regions, alpha-regions, beta-regions, spiral region, the field coil and the flexible region.

The immunoassays

Qualitative or quantitative determination of the polypeptide of the present invention in a biological sample can be carried out by any known method. The technique of using antibodies is particularly preferred for the evaluation of specific levels of polypeptides in a biological sample.

This specific recognition is the "first" antibody (polyclonal or monoclonal antibody), and the secondary detection system can be used antibodies conjugated with fluorescent substances, enzymes, or other conjugated "second" antibody. The result is immunocomplex.

In accordance with the tvii with this present invention relates to a method for the diagnosis of Aspergillus infection in certain body, incorporating the following stages:

- highlight a biological sample from the specified organism, which is assumed with Aspergillus,

interaction indicated a biological sample with the antibody of the present invention,

- assessment on the formation of immune complexes.

For release protein for Western blot analysis or dot/blot analysis, tissues can also be extracted, e.g., with urea and neutral detergent. This technique can also be applied to body fluids.

Other methods based on antibodies used for detection of gene expression of the protease are immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, monoclonal antibodies specific for the protease, can be used as immunoabsorbent and as an enzyme labeled probe for detection and quantification proteasome protein. The number proteasome protein present in the sample, can be calculated according to the amount of protein present in a standard preparation using a computer algorithm to linear regression. In another ELISA analysis can be used in two distinct specific monoclonal antibodies for detection proteasome protein in the biological fluid. In this Ana is ize one of the antibodies is used as immunoabsorbent, and the other as an enzyme labeled probe.

The above methods can be carried out, mainly in the form of "one-step" or "two-step" analysis. "One-step analysis" involves contacting proteasome protein with immobilized antibody without washing and contacting this mixture with the labeled antibody. "Two-stage analysis provides washing, and then contacting the mixture with the labeled antibody. Can also be used and other suitable methods. It is usually preferable to mobilitat one component of the analytical system on the media that enables other components of this system to come into contact with the specified component and easily stand out from the sample.

Suitable enzyme labels include, e.g., labels, derived from the oxidase group, which catalyzes the production of hydrogen peroxide by interaction with the substrate. The oxidase activity of the label can be analyzed by measuring the concentration of hydrogen peroxide formed by the interaction of the enzyme labeled antibody with a substrate.

Besides enzymes, other suitable labels include radioisotopes, such as iodine (125I121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99m TC), and fluorescent labels, such as fluorescein and rhodamine, and Biotin.

Specific binding of the test compound with the polypeptide protease can be detected, for example, in vitro, by reversible or irreversible immobilization proteasome polypeptide on a substrate, for example, on the surface of the wells of 96-well polystyrene microtiter tablet. Methods of immobilization of polypeptides and other small molecules are well known in the art. So, for example, microtiter tablets can be sensitized by protease polypeptide by adding to each well of the polypeptide in solution (usually at a concentration of from 0.05 to 1 mg/ml in a volume of 1-100 μl) and incubation of these tablets at a temperature ranging from room temperature to 37°C for 0.1 to 36 hours. Polypeptides that are not associated with the tablet can be removed by "shaking out" the excess solution from the tablet, with subsequent washing of the tablet (single or multiple) with water or buffer. Typically, the polypeptide is contained in water or buffer. The tablet then washed with buffer not containing the bound polypeptide. To block sites that communicates with the free protein tablets, these tablets are blocking a protein that is not related to the associated polypeptide. For example, suitable for this purpose is a 30 µl bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCl. Suitable substrates are substrates that contain a particular chemical cross-linking agent (e.g., plastic substrates, such as polystyrene, styrene or, for example, polypropylene substrates from Corning Costar Corp. (Cambridge, MA). If necessary, the substrate can be used granulated particle, for example, powdered agarose or granule of sepharose.

Binding of the test compounds with the polypeptides of the present invention can be detected by any known methods. For example, the immunoassay may be used a specific antibody. If necessary, this antibody can be labeled (e.g., fluorescent label or a radioisotope) and detected by the direct method (see, e.g., West & McMahon J. Cell. Biol. 74:264, 1977). Alternatively, the detection may be used "second" antibody (e.g., labeled antibody that binds to the Fc-part of an anti-N97 antibodies). In an alternative method detection proteiny polypeptide is subjected to tagging (for example, tagging proteasome polypeptide with a radioisotope, fluorophore, chromophore, or the like) and the label detected. In another way polypeptide protease produced in the form of a hybrid protein, which can be optically detected, for example, with protein, fluorescent in the green range with extra (which can be detected by UV rays). In an alternative method, the protease polypeptide can be covalently attached to the enzyme or merged with an enzyme having a detectable enzymatic activity, such as horseradish peroxidase, alkaline phosphatase, alpha-galactosidase, or glucose oxidase. Genes encoding all of these enzymes have been cloned and easily accessible to every specialist. If necessary, the hybrid protein can include an antigen, and the antigen can be detected and measured using polyclonal or monoclonal antibodies by standard methods. Suitable antigens include enzymes (such as horseradish peroxidase, alkaline phosphatase and α-galactosidase) and non-enzymatic polypeptides (e.g., serum proteins, such as BSA and globulins, as well as milk proteins, such as casein).

Epitopes, antigens and immunogenic

In another aspect, the present invention relates to a peptide or polypeptide containing epitopes part of the polypeptide of the present invention. The epitope of this part of the polypeptide is an immunogenic or antigenic epitope of the polypeptide of the present invention. The term "immunogenic epitope" is defined as the portion of the protein, which produces a humoral response in the case when the whole protein is immunogenic. Obviously, these immunogenic epitopes is limited to say the Kul multiple loci. On the other hand, the region of the protein molecules, which can bind the antibody is defined as "antigenic epitope". The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for example, H.M. Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984).

As for the selection of peptides or polypeptides bearing an antigenic epitope (i.e containing region of the protein molecule, which can bind antibody), specialists are well aware that relatively short synthetic peptides that mimic part of a protein sequence, usually capable of producing anticigarette that reacts with the partially simulated protein. Cm. for example, Sutcliffe J.G. et al., Science 219:660-666 (1984). The peptides are capable of producing reactive protein serum and is often represented in the primary sequence of a protein can be described in accordance with a simple chemical rules, and are not limited to any of the immunodominant regions of intact protein that is immunogenic epitopes)or amino - or carboxybenzene. Peptides, which are highly hydrophobic, and peptides consisting of six or less residues, are usually ineffective in inducing antibodies that bind with simulated protein, while the longer soluble peptides, particularly peptides containing prolinnova OS is atki, usually are effective. Sutcliffe et al., follow the directions above. For example, 18-20 peptides, constructed in accordance with the above guidelines and contains 8-39 residues, covering 75% sequence polypeptide chains of the ha HAI influenza virus induced antibodies that reacted with the protein HAI or intact virus; and peptides 12/12 originating from the MuLV polymerase, and the peptide 18/18, derived from the glycoprotein of rubivirus, induced antibodies that were besieging the corresponding proteins.

The peptides and polypeptides of the present invention, the bearing antigenic epitope, can be used for producing antibodies, including monoclonal antibodies that specifically bind to a polypeptide of the present invention. Thus, a large part of the hybridomas obtained by fusion of spleen cells taken from donors, immunogenic peptide bearing an antigenic epitope, usually secretes antibody reactive with the native protein. Sutcliffe et al., see above, 663. Antibodies produced antigenic epitope-bearing peptides or polypeptides can be used for detection of simulated protein, and antibodies against different peptides can be used to monitor the "fate" of various proteins precursors that undergo post-translational processing. Peptide and antibodies against the peptides can be used in various qualitative or quantitative analyses in order to determine the simulated protein, for example, in the analysis of competitive binding, as it has been shown that even short peptides (for example, consisting approximately of 9 amino acids) may contact and to substitute for the larger peptides in immunoprecipitation analyses. Cm. for example, Wilson I.A. et al., Cell 37:767-778, at 777 (1984). Antibodies against the peptides of the present invention can also be used for cleaning the simulated protein, for example, by adsorption chromatography methods well known to specialists.

Antigenic epitopes peptides and polypeptides of the present invention, constructed in accordance with the above instructions, contain a sequence of at least seven, more preferably at least nine and most preferably from about 15-30 amino acids in the amino acid sequence of the polypeptide of the present invention. However, peptides or polypeptides comprising a larger portion of the amino acid sequence of the polypeptide of the present invention containing from about 30 to about 50 amino acids, or having any length, including full amino acid sequence of the polypeptide of the present invention, are also considered epitopes peptides or polypeptides of the present invention, and can be used for the Indus is tiravanija antibodies reactive simulated protein. Preferably, the amino acid sequence epitopes peptide is chosen so that it provides good solubility in aqueous solvents (i.e., so that this sequence included a relatively hydrophilic residues and, preferably, in the main, did not contain highly hydrophobic residues); and especially preferred are sequences containing prolinnova remains.

Epitopes peptides and polypeptides of the present invention can be produced by any standard method of producing peptides or polypeptides, including recombinant methods using nucleic acid molecules of the present invention. So, for example, short epioblasma amino acid sequence can be merged with a larger acting as a carrier of a polypeptide during recombinant production and purification of recombinant and in the process of immunization to produce antibodies against the peptides.

Epitopes peptides can also be synthesized by known methods of chemical synthesis. For example, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mg of 248 different peptides, consisting of 13 residues and represent variants of a segment of polypeptide HAI, is the quiet differ by one amino acid and which have been obtained and characterized (in the analysis of binding type ELISA) for less than four weeks. Houghten R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This process of "simultaneous synthesis of multiple peptides (SMPS)was described in detail in U.S. patent No. 4631211, Houghten et al. (1986). In this procedure, a separate resin for solid-phase synthesis of various peptides are contained in a separate permeable to solvent packages that allows optimal to spend a lot of identical repetitive steps in solid-phase methods.

Manual procedure allows for simultaneously 500-1000 or more syntheses. Houghten et al., see above, 5134.

Epitopes peptides and polypeptides of the present invention are used to induce antibodies according to methods well known in the art. See, for example, Sutcliffe et al., see above; Wilson et al., see above; M. Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F.J. et al., J. Gen. Virol. 66:2347-2354 (1985).

In General, animals can be immunitary free peptide; however, the titer of antibodies against the peptide can be increased by linking the peptide to a macromolecular carrier, such as hemocyanin lymph snails (KLH) or tetanus toxoid. For instance, peptides containing cysteine, can be linked to a carrier via a linker, such as maleimidomethyl-N-hydroxysuccinimide (MBS), while other peptides may be associated with the carrier by means of a more General linking agent such as glutarimide the doctor

Animals such as rabbits, rats and mice, subjected to immunization either free or associated with a carrier peptide, for example, by intraperitoneal and/or intradermal injection of emulsions containing 10 μg of peptide or protein carrier and adjuvant's adjuvant. It may be necessary to introduce several booster injections, for example, intervals of about two weeks to get the desired titer antibodies against peptide that can be detected, for example using ELISA analysis using free peptide adsorbed on a solid surface. The titer of antibodies against the peptide in serum immunising animal can be increased by selection of antibodies against the peptide, for example, by adsorption to the peptide on a solid medium and elution of the selected antibodies according to methods well known to specialists.

Immunogenic epitopes peptides of the present invention, i.e. those parts of the protein, which produce humoral response when the whole protein is the immunogen, identify methods known in the art. For example, Geysen et al., 1984, see above, described the procedure quick competitive synthesis on solid support hundreds of peptides with a purity sufficient for the reaction in the enzyme-linked immunosorbent assay. The interaction of the synthesized peptide is in with antibodies can then be easily detected without separation from the launch vehicle. In this method, a peptide bearing immunogenic epitope of the desired protein can be identified by standard methods known in the art. For example, the localization of immunologically important epitopes in the envelope protein of a virus with a resolution of seven amino acids was determined Geysen and others, by synthesizing overlapping series of all 208 possible hexapeptides covering the entire sequence of a protein of 213 amino acids. Then was synthesized full series of peptides with substitutions, in which all 20 amino acids, in turn, were replaced at each position within the epitope, and identified the specific amino acids, indicating specificity for the reaction with the antibody. Thus, peptide analogues epitopes peptides of the present invention can be easily obtained using the above method. In U.S. patent No. 4708781, Geysen (1987)also described a method for identifying a peptide carrier immunogenic epitope of the desired protein.

Furthermore, in U.S. patent No. 5194392, Geysen (1990) described a General method of detecting or determining the sequence of monomers (amino acids or other compounds), which is a topological equivalent of the epitope (i.e. "nimotop"), which is complementary to a specific paratope (angelaswedeno site) of the desired antibodies. More specifically, in U.S. patent No. 4433092, Geysen (1989) describe the method of detecting or determining the sequence of monomers, which is a topographical equivalent of a ligand specific complementary landscapebased website needs a specific receptor. Similarly, in U.S. patent No. 5480971, Houghten, R.A. et al. (1996)related to mixtures parallelomania oligopeptides described linear parallelomania C1-C7-alkyl-oligopeptides; sets and libraries of such peptides, and methods of using such sets and libraries of oligopeptides to determine the sequence parallelomania of oligopeptides, which binds preferentially with the desired molecule acceptor. Thus, ones analogues epitopes peptides of the present invention can also be routine manufactured by these methods.

The suppression or reduction by activity

The present invention also relates to methods of producing mutant cells derived from a parent cell, which include disruption or deletion of the nucleic acid sequence that encodes a protease or a regulatory sequence that leads to the formation of mutant cell producing less protease than the parent cell.

The construction of strains which have reduced protease activity may be mainly carried out by modification or inactivation consequently the particular nucleic acid, necessary for expression by activity in the cell. Modified or inactivating the nucleic acid sequence may represent, for example, the sequence of the nucleic acid encoding the protease or its part, which plays an important role by activity, or the nucleic acid sequence may have a regulatory function required for the expression of protease from an encoding nucleic acid sequence. An example of such a regulatory or control sequence may be a promoter sequence or a functional part, i.e. the part that is sufficient to influence the expression of the protease. Other regulatory sequences for possible modifications include, but are not limited to, leader sequence, a polyadenylation sequence, propeptide sequence, the signal sequence and the site of termination.

Modification or inactivation of the nucleic acid sequence can be made by mutagenesis of this cells and selecting cells in which the capacity for production of protease was reduced or suppressed. Mutagenesis, which may be specific or nonspecific, can be carried out, for example, using the m a suitable physical or chemical mutagenesis agent, using a suitable oligonucleotide, or by CRL-generated mutagenesis of DNA sequences. In addition, mutagenesis can be performed using any combination of such mutagenesis agents. Examples of physical or chemical mutagenesis agents suitable for use in the present invention are ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethylmethanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogues. When using such agents, the mutagenesis is typically carried out by incubating mutagenicities cells in the presence of selected mutagenesis agent in suitable conditions, and selecting cells having decreased by activity or not expressing such protease activity.

Modification or inactivation of the production of the protease of the present invention can be carried out by introducing, changing or deleting one or more nucleotides in the nucleic acid sequence that encodes a protease, or a regulatory element required for the transcription or translation. So, for example, nucleotides can be incorporated or removed so that it led to the introduction of a stop codon, deletion of one hundred the t-codon or to changes in the open reading frame. Such modification or inactivation can be carried out using site-directed mutagenesis or PCR-generated mutagenesis methods known in the art.

Although, in principle, the modification can be carried out in vivo, i.e. directly in the cell expressing the modified nucleic acid sequence, preferably, the modification was carried out in vitro as described below.

An example of a suitable path inactivation or reduction of the production of protease selected the host-cell method is based on replacing the gene or interruption of the gene. For example, in the way of interrupting a gene, the nucleic acid sequence corresponding to the endogenous gene or fragment of the desired gene, subjected to in vitro mutagenesis with the production of defective nucleic acid sequence, which is then transformed into the cell host with the production of the defective gene. Using homologous recombination defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes the token that can be used for selection of transformants, in which the gene encoding the protease, has been modified or destroyed.

Alternatively, modification of alienative nucleic acid sequence, encoding the protease of the present invention, can be carried out with known methods using antisense nucleotide sequence complementary to the sequence that encodes the protease. More specifically, the production of protease by the cell may be reduced or suppressed by introducing a nucleotide sequence complementary to the nucleic acid sequence that encodes a protease that can be transcribed in the cell and is capable of gibridizatsiya with protease mRNA produced in the cell. Under conditions that allow complementary antisense nucleotide sequence to gibridizatsiya with protease mRNA, the number of transmitted thus protease is reduced or suppressed.

In this case, preferably, the cell is modified by the methods of the present invention, was of microbial origin, for example, was derived from a fungal strain, suitable for producing the desired protein product, which is homologous or heterologous to the cell.

In addition, the present invention relates to mutant cell derived from a parent cell and including disruption or a deletion in a nucleic acid sequence that encodes a protease, or a regulatory sequence, the result is it this mutant cell produces less of the protease, than the parent cell.

Thus obtained deficient in protease mutant cells are particularly suitable as host cells used for the expression of homologous and/or heterologous polypeptides. Therefore, the present invention relates to methods for production of homologous or heterologous polypeptide, comprising (a) cultivating the mutant cell under conditions suitable for production of the polypeptide, and (b) isolation of the polypeptide. In the context of the present description used herein, the term "heterologous polypeptide" means a polypeptide that is not native to the host cell; native protein, which had been modified to change the native sequence, or a native protein, the expression of which was quantitatively altered by modification of the host cell by methods of recombinant DNA.

The methods of the present invention for producing essentially not containing protease product, are of particular interest for the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes. Deficient in protease cells can also be used for expression of heterologous proteins of interest for the food or pharmaceutical industry.

The use of proteases in industry the industrial production

The present invention also relates to the use of the protease of the present invention in some ways, used in industry and in the pharmaceutical industry. Despite years of experience in applying these methods, the protease of the present invention has several significant advantages compared to the enzymes used in the present time. Depending on the particular application, such advantages can be factors such as lower production costs, higher specificity to the substrate, a lower antigenicity, less unwanted side effects, higher yields when producing in a suitable microorganism, more suitable ranges of pH and temperature, the best taste of the final product, as well as higher quality of food and its kosher.

In large-scale industrial production of feed or food proteolytic enzymes are commonly used to improve parameters such as the solubility of the protein, the yields of products extraction, viscosity or taste, texture, nutritional value, minimization of antigenicity or levels of harmful factors, color, or functionality; as well as to improve process parameters, such as filterability raw protein. For these applications protein materials can be policenet animal or plant and its examples include vegetable proteins such as soy protein, wheat gluten, protein, rapeseed, pea protein, alfalfa protein, protein, sunflower protein, legumes, fiber, cotton or sesame protein, corn protein, barley protein sorghum, potato protein, rice protein, coffee and proteins animal proteins such as milk protein (e.g. casein, whey protein), egg protein, fish protein, meat protein, including gelatin, collagen, blood protein (e.g., hemoglobin), squirrel hair, feathers and fish meal.

An important property of the protease of the present invention lies in the fact that they operate at optimum pH range and optimum temperature, which is ideal for a variety of purposes. So, for example, to minimize the risk of microbial infections, many large-scale processes are preferably carried out at relatively high temperatures of processing, i.e. at 50°C or higher. Some of the protease of the present invention satisfy these requirements, but they do not have excessive thermal stability, such that resisted all attempts to inactivate this enzyme by additional heat treatment. The last of these distinctive features allows the production, which gives the final products, nesadurai residual proteolytic activity. Similarly, many feed and food products have low pH values, and therefore their processing, it is preferable to use protease with optimal acid pH or nearly neutral pH. The protease of the present invention also satisfies these requirements.

The specificity of endoprotease usually determined by the preferred cleavage relationships between carboxyla amino acid residue in position P1 and the amino group of this residue in position P1', respectively. Preference may be determined predominantly either P1 (for example, positively charged residues in substrates for trypsin), or P1' (for example, hydrophobic residues at the cleavage by thermolysin), or P1 and P2 (for example, a specific splitting ties between the two positively charged residues of the serine endoproteases the medulla of the adrenal gland). In some cases, more distant residues can determine the preference of cleavage, for example, P2 is preferred for strep peptidases A. it is Known that some residues have a negative impact on cleavage, and is also well known that prolinnova communication position P1' are resistant to the action of many enzymes. Most of endoprotease split predominantly either in the hydrophobic environment or close to the negative the positive charged residues. So, for example, used in industry endoprotease, such as chymotrypsin (from bovine pancreas) or subtilisin, neutral metalloendopeptidases or thermolysin (derived from Bacillus species), have a tendency to splitting on the area "behind" hydrophobic amino acids such as Phe, -Leu-Tyr. Other used in industry endoprotease are trypsin (from bovine pancreas), splitting mostly behind-Arg-Lys, and papain (a complex mixture of various enzymes, including protease, derived from papaya fruit), splitting mostly behind-Arg. In contrast, the peptide bond formed by small residues, such as Ala, Gly, Ser, Thr, and Ile and Pro are poor substrates (B. Keil et al., Protein Seq Data Anal. (1993), 5; 401-407). This situation creates difficulties for the pharmaceutical industry, namely in the production of food and beverages, agriculture and chemical industry. The protease of the present invention has an unusual preference in the splitting.

Ectopeptidases act only near the ends of polypeptide chains. Such action of peptidases at the free N-Terminus leads to the release of a single amino acid residue (so-called aminopeptidase) or dipeptide or trip is putida (the so-called dipeptidylpeptidase and tripeptidylpeptidase). Such action of peptidases at the free-end leads to the release of a single amino acid residue (so-called carboxypeptidase) or dipeptide (so-called peptidyl-dipeptidase). In its catalytic mechanism of carboxypeptidase can be divided into three groups, namely serine carboxypeptidase type, metallocarboxypeptidase and cysteine carboxypeptidase type. Other ectopeptidases are specific for dipeptides (the so-called dipeptidase) or is able to cleave peptide bonds non-bonds of the alpha-carboxyl or alpha-amino groups (the so-called omega-peptidases). Examples of such new omega peptidases are Pyroglutamate and acetamidoacrylate identified in the present invention (see table 1, genes 18 and 45, respectively).

Typical examples of industrial application, which depends on the use of clean endoprotease, and in which the protease of the present invention may be supposed to possess excellent properties are processing materials of vegetable or animal origin. These processing stages can be targeted for modification of a large array of characteristics of the raw material or (partially) purified protein fraction. For example, these processing stages can be carried out for m is kimitachi solubility product, filterability, the ability to selection of the outputs of the protein after extraction and its digestibility or to minimize toxicity, strange taste and viscosity. In addition, such processing may be directed to the change of physico-chemical properties of raw material or purified (or partially purified) protein. These advantages can be used not only in cases when endoprotease of the present invention is used to facilitate processing in industrial production, but also in cases when it is used as an active enzyme component in animal feed. In particular, endoprotease of the present invention can be used as a bread improver in the baking industry, for example, to retard the staling of bread or to reduce the viscosity of the test. Or endoprotease can be used in the brewing and wine industries to prevent or to minimize the formation of undesirable protein haze. Alternatively, it can be used in the brewing industry to optimize the yields of extraction of protein grain that is used to obtain the wort. In addition, it can also be advantageously used in the dairy industry as an agent for the coagulation of milk with improved properties and or to optimize texturing, flushing or coagulation properties of milk components. Another application of the new protease of the present invention in the dairy industry is its use for the preparation of modified fermented cheeses (ISF).

In addition, various protein substrates can be subjected to endoprotease of the present invention, usually in combination with other proteolytic enzymes to obtain hydrolysates for use in medical and non-medical purposes. Unexpectedly, it was found that endoprotease of the present invention is effective for the complete hydrolysis of the protein substrate, such that it completely hydrolyzed even the parts that are resistant to the protease, and, in addition, it was unexpectedly discovered that this endoprotease also has activity aimed at minimizing the allergenicity of the final hydrolysate or to suppress the formation of bitter taste.

More specifically, endoprotease of the present invention differs in its preferences in the unusual splitting of peptide bonds of proteins, especially in amino acid residues of a small size, such as Ala, Gly, Ser and Thr, or in the residues Ile and Pro at position P1 or P1' (B. Keil et al., Protein Seq Data Anal. (1993) 5; 401-407). The resulting fraction is elkovich raw materials, which turned out to be resistant to hydrolysis after using the known endoprotease, can be dissolved and hydrolysed using endoprotease of the present invention. Non-limiting examples of such resistant to protease fractions are the so-called extensive in plant materials, collagen, gelatin, and specific milk components in materials of animal origin.

Various food products such as soybeans contain trypsin inhibitors. These proteins inhibit Triesenberg activity in the gastrointestinal tract, for example, in pigs and poultry. This inhibiting trypsin activity leads to poor digestibility of the protein in these animals, and therefore to the increase of waste and increased material costs. This problem can be partially solved by roasting soybeans at high temperatures. In soybeans were identified inhibitors of trypsin two different types, namely, trypsin inhibitors of the Bowman-Tag and trypsin inhibitors Marten.

The present invention also relates to an alternative path of destruction trypsin-inhibiting activity at toasting, which is provided by the cysteine proteases (EC 3.4.22, table 1), is able to cleave the peptide bond leucine 176 - aspartate 177 near the carboxyl end of inhibit the RA trypsin Marten (as described by Wilson (1988), CRC Critical Reviews in Biotechnology 8(3):197-216). This path leads to inactivation of the specified trypsin inhibitor in soy. It was unexpectedly found that cysteine protease, secreted by the fungi Aspergillus niger, meet these criteria much better than similar enzymes, derived from other organisms.

Protease is also widely used in the production of cheese. In the manufacture of cheese milk for cheese making needs to be folded, in order to separate the cheese substance from whey, for example, casein. Have been described, some enzymes of the coagulation of milk, also known as coagulants, and such enzymes are (cow) chymosin, bovine pepsin, porcine pepsin, as well as microbial enzymes, such as protease Rhizomucor miehei, protease Rhizomucor pusillus and protease Cryptonectria parasitica. Chymosin can be obtained from the stomach of a calf, but it can also be produced by microbes, for example, Kluyveromyces lactis. All these enzymes are characterized by the fact that they possess a specificity for peptide bond between residue 105 (phenylalanine) and residue 106 (methionine) or communication related to communication in casein. This means that when using such enzymes in the manufacture of cheese, casein is cleaved at the junction between the para-casein and macropeptide part, called glycomacropeptide (GMP) and carrying negative charges. If micropa the ID diffuses into the whey, its stabilizing effect on the solubility of the casein micelles is lost, and these casein micelles can start to aggregated after sufficient hydrolysis of the Kappa-casein. A detailed description of the enzymatic coagulation of milk can be found, for example, D.G. Dalgleish, Advanced Dairy Chemistry, vol.1 ed. by P.F. Fox, Elsevier, London, 1992.

Currently used coagulants allow to obtain a sufficiently high yield of cheese, however, it should be noted that this is due to the large volumes of produced cheese, and increase output by approximately one-tenth of a percent can make a tangible economic benefits. Therefore, at present there is an extreme need for coagulants, which gives the increase (even small) exit.

Coagulants are characterized by their high specificity to the substrate, which is usually dependent on pH and temperature. In conventional processes for the production of cheese, the pH may vary from the initial value of 6.3 to lower values in the range of 4.5 to 5.5, and the final pH value depends on the conditions used in the process of making cheese. Some coagulants are more susceptible to changes in pH than others. So, for example, protease Rhizomucor pusillus is more sensitive to changes in pH than chitosan. In addition to pH, the specificity of the protease can also be influenced by other parameters is s, such as temperature and water content. It is well known that the majority of coagulants detect the change of specificity to the substrate with a change in pH, which leads to changes in proteolytic activity in the final stages of making cheese. Also very well known that the coagulants differ according to the degree of proteolysis of casein; and, in addition, they can detect differences in the types of peptides produced in the process of proteolysis. They are important factors in the process of ripening of cheese and can affect the properties of the cheese, such as taste, odor and texture. In some cases, coagulants produce undesirable effects such as the appearance of bitter taste, called peptides, or strange taste. In addition, changes in proteolytic specificity may lead to reduced output. Pepsin, a well-known component that is present in many preparations of bovine chymosin is an example of a protease, which gives lower yields and the worst taste effects than pure chymosin. So you need to get coagulants, which would give the cheese a new and improved consistency and taste. Such new coagulants will lead to accelerated formation of taste and profiles consistency associated with the ripening of cheese that can provide tangible economic the th benefit.

It is well known that free amino acids are very important for the formation of taste and smell. In particular, the amino acids leucine, phenylalanine, methionine and valine play an important role in the formation of components of a typical cheese taste and smell. The conversion of free amino acids occurs through the fermentation of microorganisms, which is added in the process of cheese making connections, forming the desired smell and taste, such as methanediol, dimethyl disulfide, methylpropanoate acid and methylpropanal. Ectopeptidases play an important role in the formation of free amino acids. However, they can only be effective if they work together with endoproteases appropriate specificity. In the preparation of cheeses can be used in appropriate combination Exo - and endopeptidase, the result can be obtained cheeses with new and improved taste.

The enzymes of the present invention can be used for hydrolysis of protein materials of animal origin such as whole milk, separated milk, casein, whey protein or a mixture of casein and whey protein. Such a mixture of casein and whey protein can be used, for example, in the relationship corresponding to the relationship found is Emim in human milk. In addition, a mixture of enzymes of the present invention can be used for hydrolysis of protein materials of vegetable origin, such as, for example, wheat gluten, malt or necrotomy barley or other cereals used in the production of beer, soy milk, concentrates or isolates, protein maize, concentrates or isolates and rice proteins.

With regard to large-scale industrial production, some applications based on the use of only endoprotease, whereas other applications based on combinations of endoprotease and ecoprocess. Typical examples of processes that depend on the use of clean endoprotease, in which the protease of the present invention can have excellent productivity, are processes such as the processing of soy protein, pea or grain to minimize viscosity or optimization of foam or other physico-chemical properties; the use of bread improvers in bakery industry, which also contributes to reducing the viscosity of the dough; use of additives in the brewing and wine industries, which helps prevent protein haze or optimize yield after extraction of grain; the use of feed additives in the production of biocarbon to the / establishment, which enhances intestinal absorption or modulation of microbial activity in the gut; the use of additives in the dairy industry, which contributes to the optimization of coagulation, foaming or coagulation properties of milk components. In addition, for specific market segments, dairy or soy proteins, or collagen is subjected to the action of proteases in order to produce the so-called protein hydrolysates. Although the hydrolysates of these proteins, mainly used for the manufacture of baby food and food for hospitalized patients, but they can be also designed for individuals who do not need medical attention, such as athletes or people who are on a diet for weight loss, and are subject to rapidly growing demand in the market. In all these applications hydrolysates of proteins possess very attractive properties as reduced allergenicity, improved digestibility in the gastrointestinal tract, low level chemical decomposition of the desired amino acids such as glutamine and cysteine, and, finally, the absence of the protein precipitate in acidic beverages during prolonged storage. All these advantages can be combined, if the hydrolysate is a mixture of di - and tripeptides. However, all modern commercially available hydrolysates obtained using combinations of the number of the x endoprotease. This last method gives non-uniform and incomplete decomposition of protein. To obtain a desired mixture of di - and tripeptides it would be ideal to use in the hydrolysis of a combination of different di - and tripeptidylpeptidase. Unfortunately, there were only few enzymes food purposes and microorganisms suitable for use in industry, not to mention enzymes, is actually available for use in industry. In accordance with the present invention, several widely used di - and tripeptidylpeptidase can be obtained cost-effective way and in a relatively pure form. Preferred are di - and tripeptidylpeptidase that have low selectivity with respect to biodegradable substrate, that is, have a preference only to the splitting of the minimum number of amino acid residues. Preferred are combinations of such di - and tripeptidylpeptidase that hydrolyzing a high percentage of natural peptide bonds. Despite this high level of activity in relation to natural peptide linkages, complete hydrolysis of all amino acids depends on the nature of di - and tripeptidylpeptidase. Preferred are di - and tripeptidylpeptidase that have optimal activity at pH 4-8 and have adequate thermal stability. Hell is a cotton stability means, that at least 40%, preferably at least 60%, more preferably 70-100% of the initial hydrolytic activity is retained after heating the enzyme with substrate for 1 hour at 50°C.

Although the process for the efficient production of mixtures of di - or tripeptides, depends on the availability of the enzymes of the present invention, however, the initial enzyme for incubation with the substrate protein is usually endoprotease. The preferred endoprotease with a wide range endopeptidase activity suitable for this case is, for example, subtilisin (Delvolase from DSM), neutral metalloprotease (neutrase from NOVO) or thermolysin (termbase from Daiwa Kasei)used in conditions close to neutral, and pepsin or aspergillosis (for example, Sumizyme AP from Shin Nihon, Japan)used in acidic conditions. The purpose of this initial splitting is to increase the solubility, lower viscosity and lower termousadke ability of a mixture of water and protein. In addition, this pre-processing endonuclease is important to create sufficient starting points for di - and tripeptidylpeptidase and leads to the acceleration of the process of formation of di - or Tripeptide. The protease used to eliminate the bitterness of the hydrolyzate may be, but not necessarily, included in this stud is Y. this method or later, together with di - and tripeptidylpeptidase.

The main purpose of the use of hydrolysates is to minimize the allergenicity of the product or stimulation digestibility in the gastrointestinal tract. When producing such hydrolysates using dipeptidyl and tripeptidylpeptidase is particularly important and is an effective way of producing hydrolysates.

Another application in the production of food and feed mainly based on combinations of one or more endoprotease with one or more ekzoprotezov. Such combinations of endoprotease with ecoprotect commonly used in industry to improve parameters such as taste and color of the final product. The reason for this is that the formation of taste and color depends mainly on the presence of free amino acids. Free amino acids can be produced not only by proteases such as carboxypeptidase and amino peptidases, but also peptidylarginine. When combined with endoprotease or even with dipeptidyl or tripeptidylpeptidase, carboxypeptidase, amino peptidases and patibilities can produce a greater amount of free amino acids over a shorter period of time. However, in all these processes uncontrolled release of amino acids or even nabalco the s components should be prevented to minimize undesirable side reactions.

While free amino acids themselves can produce a range of taste sensations, and these taste sensations are the most important (bitter, sweet, sour and umami), but to distinguish between these tastes very high concentration of amino acids. Despite the high threshold value, free amino acids can create significant sensory effects at lower concentrations due to a number of flavor-enhancing mechanisms. One of these mechanisms is the combination of free amino acids with sugars in the so-called Maillard reactions. Compared with the free amino acids these Maillard products with very complex bouquet of taste and smell can be produced with the threshold values that are several orders of magnitude lower than those produced by free amino acids. The Maillard products can be formed at elevated temperatures, usually during cooking, baking or frying in the preparation of food or feed. During this treatment develops color and a large bouquet of aromas. In these reactions, in the first stage, the amino groups react with reducing compounds, which ultimately leads to the implementation of a whole cascade of reactions. The amino compounds, mainly present in food or feed, are free amino acids, which are released from the raw protein by different protease and necessary restorative compounds are mainly reducing sugar. It follows that to minimize foreign flavor, which is generated during the subsequent stages of heating, such as during spray drying or sterilization, it is necessary to prevent unwanted release of amino acids and sugars during processing of raw materials. This once again indicates that it is desirable to use the enzymes of the present invention, which have a high degree of purity and low cost.

In addition to Maillard reactions, amino acids may also be important chemical transformations at room temperature. Later types of transformation are processes dependent enzymes, and processes common to all fermented foods, such as maturation of beer, yogurt and cheese, as well as the processes of maturation of meat and wine. In these fermentation processes free amino acids are released from the raw material used by proteases or proteolytic enzymes present in the used raw material or microbial yeast. Then in the phase of ripening free amino acids under the action of the microbe is Oh metabolic activity into their derivatives, with increased sensory properties. So, for example, L-leucine, L-isoleucine and L-valine at beer fermentation contribute to the formation of valuable fusel alcohols such as amyl alcohol and Isobutanol. Similarly, it was found that cheese volatile substances, such as methanethiol and dimethyl disulfide, indicate the presence in the cheese methionine, and methylpropanoate acid and methylpropanal - the presence of valine. And finally, the free amino acid glutamate may give effect to enhance a pleasant spicy taste as a result of its synergistic action with the products of the cleavage of RNA, the so-called 5'-ribonucleotides. It is known that when combined with the appropriate concentrations of 5'-ribonucleotides such as 5'-IMP and 5'-GMP, the threshold detection of the formation of taste "umami"generated by glutamate decreases by almost two orders of magnitude.

To obtain explicit and well-defined taste effects in all of these procedures protein substrates must be hydrolyzed using a combination of endo - and ecoprocess, where at least one endo - or ectoprocta, and preferably, as endoprotease and ectoprocta are cleaned and preferably, selective with respect to a particular series of amino acids or mainly split the preferred amino acids Such preferred proteases are characterized by a high selectivity with respect to amino acid sequences, which can be split by enzymes belonging to the category of Aspergillus, known as the "maturity" and representing a particular value.

In addition to the use in the food industry and in food production, protease also typically used in chemical and pharmaceutical industries, as well as in the production of diagnostic reagents and personal hygiene. In the manufacture of personal hygiene, proteases are used to obtain peptides that add various products to improve properties such as sensitivity and the glossiness of the skin, or to protect the skin. In addition, there is a new trend to direct local application of proteases. Similarly, this enzyme can be used in the leather industry, and the main purpose of this application is to clean, remove the wool from the skin and softening the skin.

In the chemical and pharmaceutical industries protease considered as an important tool for the production of valuable ingredients or intermediates. In these industries protease are used not only because of their hydrolytic activity, but also because of their ability to the synthesis of peptides from natural or unnatural amino acids. The last of the possible applications has been clearly demonstrated is the opportunity to synthesize aspartame of the structural elements, consisting of amino acids, using endoprotease, such as thermolysin.

In contrast to the conditions used in the production of food and feed, to implement the desired chemical transformations, are very important factors are stereo - and regioselectivity of proteases, although in this case may require non-standard reaction conditions. Typical examples of the application of proteases in this industry is the use of endoprotease, aminopeptidase and carboxypeptidase in the production of various intermediate compounds in order to obtain drugs, such as insulin, antibiotics, renin and ACE-inhibitors. The review of such applications are presented in Industrial Biotransformations, A. Liese, K. Seelbach, C. Wandrey, Wiley-VCH; ISBH 3-527-30094-5.

With regard to the desired specificity, stereo and regioselectivity, an important advantage of the protease of the present invention is the absence of side effects and resistance to unusual reaction conditions, such as high solvent concentration, and increased productivity.

From the pharmaceutical point of view, the role of proteases are presented in a wide range of works Martindale, "The Extra Pharmacopoeia" (Pharmaceutical Press, London, UK). In addition, the important role of specific proteases in the regulation of all kinds of biological processes is confirmed by the fact that many of the hormones become active only after processing, mainly inactive molecules predecessor specified protease with very high specificity. Inhibitors that are active against certain categories such specific proteases were used in the development of all types of new medicines. Therefore, new and effective protease inhibitors can be identified using the sequences described here.

A full description of each cited here document is entered into this application by reference.

ES.-
Table 1
Room SEQ IDThe function of the encoded proteinEC-number
GenecDNAProtein
158115Pepsin And3ES
259116MetalloproteaseES
360117AcylaminoacidsES
461118TripeptidylpeptidaseES.-
562119 The serine carboxypeptidaseES
663120The serine endoproteaseES.-
764121The carboxypeptidase YES
865122Aspergillosis II - d.ES
966123TripeptidylpeptidaseES
1067124TripeptidylpeptidaseES
1168125Aspergillosis II - d.ES
1269126TripeptidylpeptidaseES
1370127MetalloproteaseES.-
1471128Aspergillomas IES
1572129The pepsinogen EES
1673130Aspergillomas I - d.ES
1774131TSA is gillepsy II ES
1875132Pyro-Glu-peptidaseES
1976133The dipeptidyl peptidaseES
2077134Secretiruema aminopeptidaseES
2178135Alkaline D-peptidaseES
2279136The carboxypeptidaseES
2380137The carboxypeptidaseES
2481138The carboxypeptidase IIES
2582139Aspartic proteaseES.-
2683140TripeptidylpeptidaseES
2784141The carboxypeptidaseES
2885142Cysteine proteinaseES.-
2986143Metallocarboxypeptidase
3087144Subtilisin - d.ES
3188145The carboxypeptidase YES
3289146MetalloproteaseES.-
3390147The carboxypeptidase YES
3491148MetalloproteaseES.-
3592149TripeptidylpeptidaseES
3693150Aspartic proteaseES
3794151Aspartic proteaseES
3895152Pepsin And3ES
3996153Aspartic proteaseES
4097154Aspartic proteaseES
4198155KEHES
42 99156Serine proteaseES.-
43100157GlutamyltranspeptidaseES
44101158Aspergillosis II - d.ES
45102159AcylaminoacidsES
46103160TripeptidylpeptidaseES.-
47104161The serine carboxypeptidaseES
48105162Gly-X carboxypeptidaseES
49106163Aspartic proteinaseES.-
50107164TripeptidylpeptidaseES
51108165The carboxypeptidase-IES
52109166The serine carboxypeptidaseES
53110167The serine carboxypeptidaseES
54111168Secretiruema aminopeptidaseES
55112169PolyangiitisES
56113170Aspergillomas I - d.ES
57114171AminopeptidaseES.-

EXAMPLES

Example 1

Analysis of proteolytic activity and specificity

The specificity of the prosthesis can be investigated using a variety of peptide substrates. Synthetic substrates are widely used for the detection of proteolytic enzymes in screening, fermentation, process selection for analysis of enzyme activity, for determination of the concentration of enzyme to study the specificity or to study the interaction with inhibitors. Peptide p-nitroanilide preferably used for analysis by activity, and because such activity can be continuously traced, it allows kinetic measurements. Monitoring cleavage of the peptide p-nitroanilide can be carried out by measuring the increase in absorption at 410 nm after the release of 4-nitroanilide. Parani rosaniline substrates are typically used for serine and cysteine proteases. In addition, use thioethers peptides and derivatives of 7-amino-p-methylcoumarine peptides. The thioethers of the peptides are highly sensitive substrates for serine proteases and metalloproteases, which have a relatively high rate of functional cycle, because the thioester bond is cleaved more easily than amide bond. Cleavage of thioethers can be seen using a thiol reagent such as 4,4-zitieren (324 nm) or 5,5-dithiobis-2-nitrobenzoic acid (405 nm). The same high speed functional cycle is usually observed in the cleavage of ester groups in comparison with the speed of the cleavage of amide bonds. The most well-known substrates for analysis esterase activity of proteases are derivatives of p-NITROPHENOL. The release of p-NITROPHENOL can be seen at different wavelengths depending on the pH; for example, at an almost neutral pH is used wavelength of 340 nm and at pH above 9 monitoring carried out at about 405 nm. In addition, monitoring of the hydrolysis of esters can also be carried out by titration using equipment pH-stat. In the case of sound measurements esterase activity can be used pH-sensitive dyes.

Alternatively, the peptides can the be attached to the fluorescent leaving group. Proteolysis accompanied by an increase in fluorescence monitoring at appropriate wavelengths. The most frequently used peptidyl-2-naphthylamide and peptidyl-4-methyl-7-kumarihamy. The release, for example, 7-amino-4-methylcoumarin was measured at a wavelength of excitation 350 nm and emission wavelength of 460 nm. The use of 7-amino-4-triptorelin has the advantage that it is withdrawing group is a chromogenic (absorption at 380 nm), and fluorogenic (vozbuzhenie 400 nm, emission at 505 nm). If you want on both sides of the split connection was present amino acid, it may be useful to introduce the group to fluorescence quenching. General properties of such substrates is that in them the fluorescent donor group is separated from the acceptor group of the peptide sequence that acts as a quencher of fluorescence. Cleavage of the peptide bond between the quenching group and the fluorophore can lead to a significant increase in fluorescence. It has been described several pairs of "donor-acceptor", including the o-aminobenzoic acid (Abz), used as a donor, and 2,4-dinitrophenyl (Dnp)is used as acceptor, or 5-[(2'-amino-ethyl)amino]naphtalenesulfonic acid (EDANS), used as a donor, and 4-[[4'-(dimethylamino]phenyl]azo]benthological (DABCYL), used as acceptor. Abz/EDDnp is the most suitable couple of "donor-acceptor", because after complete hydrolysis, the fluorescence intensity increases in 7-100 times, and the absorption spectrum EDDnp not change with pH. In addition, the peptide sequence can contain up to 10 residues, but it loses its quenching action. Increasing the size of the connecting peptide of the provisions of this split may be less specific. Therefore, in addition to establishing event proteolysis may be necessary to conduct additional analysis of these products. This can be done through the analysis and selection produced peptides using HPLC and determination of the amino acid sequences of these fragments. In addition, the peptide composition of the hydrolyzate can be directly analyzed by combined method using HPLC/mass spectroscopy.

In addition to using the peptide sequence, to study the specificity of the protease can also be used libraries of synthetic peptides. Peptides were synthesized using solid-phase synthesis of randomized or pourandarjani way. For example, Meldal et al. (PNAS USA 91, 3314, 1994) reported receiving collection substrates for the por is teas by attaching N-Lys(Abz)-resin, elongation resin peptides with a length of up to 6 amino acids and, finally, joining Tyr(NO2to these peptides. Each granule resin has a unique sequence, and after treatment with proteases the most sensitive fluorescent granules become as the release Tyr(NO2)-containing peptide. Sequence analysis of these peptides on their sensitivity will provide information on the specificity of the protease.

Proteasa activity is usually expressed in units. Generally speaking, "a standard international unit (IU) is defined as the amount of enzyme which under certain conditions takes one micromoles of substrate per minute. In particular, with regard to proteases, the IU must comply with the hydrolysis of one micromole peptide bond in a minute. However, in the case of protease, deviations in the determination of its units from the international standard is the rule rather than the exception. In the case of peptides that specifically split on one communication, computation IU is narrow procedure, and for protein substrates, where the cleavage of the protease can be carried out in different positions and in different degrees, may be adopted another definition that differs substantially from the standard. In addition to the definitions of activity units, any e is speriment hydrolysis requires an adequate description of the conditions, at which carry out the measurement of these units. Such conditions include, for example, the substrate concentration, the enzyme-substrate", pH and temperature. Typical analyses to determine the specific activity of proteases include the use of protein substrate, such as, for example, denatured hemoglobin, insulin, or casein. The polypeptide substrate is cleaved by the protease under fixed conditions for a specific period of time. Unsplit and large polypeptides precipitated using TCA and TCA-soluble product is evaluated by measuring the optical density at 220 or 280 nm, or by titration of soluble peptides foliowym reagent, ninhydrin, fluoro-2,4-dinitrobenzene/danielgordon, a method using TNBS or fluorescein. Instead of labeling the product after hydrolysis can also be used polypeptide substrates that were already marked by specific dyes or fluorophores, such as fluorescein. In addition to the standard methods of amino acid analysis can be performed using standard laboratory analyzers. To estimate the size distribution of peptides generated by protease can be carried out experiments using gel chromatography. In addition, to better highlight ipov peptides generated by the protease, can be used HPLC using reversed-phase technique. The progress of hydrolysis of protein substrates are usually estimated by the degree of hydrolysis or DH. When using the pH-stat to track the progress of hydrolysis DH can be determined based on the absorption of the base hydrolysis (Enzymatic Hydrolysis of Food Protein, J. Adler-Nissen, 1986, Elsevier Applied Science Publishers LTD). DH means a variety of useful functional properties of the hydrolysate, such as solubility, emulsifying capacity, foaming capacity and stability to foaming, the increase in volume when whipping, organoleptic quality. In addition, an important parameter in food hydrolysates is the taste. Bitterness can be a major problem when using protein hydrolysates. The completion of the hydrolysis reaction can be carried out by changing the pH, thermoinactivation and application of denaturing agents, such as LTO, acetonitrile, etc.

The polypeptides presented in table 1, were expressed and at least partially purified by standard methods known in the art. These polypeptides were analyzed, at least one of the methods described above, and found that they have the activities listed in table 1.

Example 2

A direct relationship definition kcat/Km for substrates of Protea the s

For monitoring enzymatic activity in the cleaning process, to determine the concentration of the substrate, to define constants of inhibition or to study the specificity of the substrate were used synthetic substrates. The relationship definition kcat/Km allows to measure the specificity to the substrate. It also allows comparison of specificdate different substrates for the same enzyme or the comparison of the velocities of hydrolysis of various enzymes that cleave the same substrate. This ratio represents the rate constant expressed in units of the second order, and is defined as 1/(concentration · the time). The substrates with respect to kcat/Km in the range of 10.5-10.6 M-1·s-1, considered to be very good substrates, that is, with good affinity and rapid functional cycle. However, some substrates can be very specific, i.e. to have the values of kcat/Km, constituting approximately 10.4 M-1·s-1.

The ratio kcat/Km can be calculated after determining the individual parameters. In this case, Km and Vm can be determined from various linear curves (constructed, for example, by the method of Hans or Cornish-Bowden) or by the method of nonlinear regression. It is known that Vm=kcat·Et (where Et denotes the final concentration of active EN zymes is a), consequently kcat=Vm/Et. The relationship definition kcat/Km above described method may be difficult if there is inhibition of the product or substrate or if the substrate is deposited at high concentration. However, you can get the exact value of the ratio of kcat/Km under the reaction conditions of the first order, that is, when the substrate concentration is significant below the estimated Km. Under these conditions, the equation of Michaelis-Menten: v=(Vm·S)/(Km+S) has the form:

v=(Vm·S)/Km, if S ≪ Km

or v=(Vm/Km)·S= kobs·S=-dS/dt

and after integration get: lnS=-kobs·t+InSo, where So denotes the initial concentration of the substrate, and S denotes the substrate concentration at a given time. The speed is proportional to the concentration of the substrate. In other words, the hydrolysis of the substrate proceeds in accordance with a first order reaction, where kobs is the rate constant of the first order: kobs=Vm/Km=(kcat·Et)/Km, if Vm=kcat·Et. Continuous registration of the hydrolysis of the substrate allows for the graphical determination of kobs on curve lnS from time to time. The ratio of kcat/Km is simply determined on the basis of kobs, provided that a known concentration of active enzyme:

kcat/Km=kobs/Et.

Analysis method: using the initial concentration of the primary substrate constituting much less than the estimated Km, and a low concentration of the enzyme allows you to register Ho the hydrolysis of the substrate. On this basis, to generate the product get the curve of the first order. After complete hydrolysis of the substrate of the optical density or fluorescence units) of the product can be accurately determined on the basis of So, for Pt=So·kobs is determined from the curve lnS from time to time, or alternatively, it can be determined using software selection curve points (Enzfitter, SigmaPlot...).

NB: it Should be remembered that it is necessary to calculate the substrate concentration for a given point in time based on the concentration of the product (S=So-R), since the dependence of R on time gives the correct values of kobs (dP/dt=kobs·S not integrated in the same way).

Alternatively, it is possible to measure the successive values of t1/2(half) on the basis of the curve of production of the product in the reaction of the first order:

t1/2=ln2/kobs=0,693/kobs, and kobs=0,693/t1/2

Using this method allows you to control the true attenuation of the first order (identical values for successive values of t1/2).

Example 3

Inactivation of protease genes in Aspergillus

The most appropriate way of inactivation of protease genes in the genome of Aspergillus is the technique of gene replacement (so-called "single stage disrupted gene"). The principles of this technique are described Rothstein J., Meth. Enzymol. 101, p202, 1983. Basically, this technique osnovana homologous recombination transformed DNA fragments from the genomic DNA of the fungal cell. By double crossover, inactivating the gene is replaced by the (partial) DNA fragment, which is transformed cell. Preferably, the transforming DNA fragment contains a selective marker gene of Aspergillus niger. Modification of DNA and generation of inactivating design carried out mainly using the General methods of molecular biology. First, genomic DNA isolated from strains of Aspergillus niger, which is then used for the inactivation of the protease gene. Genomic DNA A.niger can be allocated any of the methods described, for example, by the method described Graaff et al. (1988) Curr. Genet. 13, 315-321, and by methods well known in the art. This genomic DNA is used as template for amplification of flanking regions protease gene by polymerase chain reaction (PCR; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). The term "flanking region" means a non-coding region located above and below inactivating protease gene. Preferably, each flanking region had a length of more than 1.0 TPN

For the implementation of PCR amplification of each flanking region of the primers used two single-stranded DNA oligonucleotide. For 5'-flanking region used one primer is homologous to a DNA sequence that is located higher in chirouse codon coding sequence of the gene of the protease. Preferably, the homologous region was localized at the site located more than 1.0 TPN above the site of translation initiation. The second primer was homologous complementary and inverted DNA sequence located immediately above the coding sequence of the gene of the protease.

For the 3'flanking region used one primer, which was homologous to a DNA sequence that is located directly below the coding sequence of the gene of the protease. The second primer was homologous complementary and inverted DNA sequence, localized, preferably, at the site located more than 1.0 TPN below the coding sequence of the gene of the protease.

The DNA sequence of the primers and homologous to the genome of A.niger, must have a length of at least 15 nucleotides, and preferably more than 18 nucleotides. Basically, all the primers should contain a DNA sequence encoding a site of recognition for appropriate restrictively enzymes located above sequence homologous to the genome of A.niger. These additional recognition sites to facilitate the cloning process.

Primers and genomic DNA A.niger used in a PCR reaction under conditions known in the art. The temperature of annealing of primers mo is et to be calculated for a portion of the DNA sequence, which is homologous to the genome of A.niger. Both fragments containing 5'-flanking region and 3'-flanking region was cloned into a vector that can be replicated in E. coli using the General methods of molecular biology. Then gene, which can be used as a selective marker in Aspergillus niger was cloned between the two flanking regions. Typically, the marker gene is under the control of a promoter that regulates the expression of A.niger, and preferably, the endogenous promoter A.niger. The orientation of the insertion of the marker gene, preferably coincides with the orientation of the original protease gene. End inactivating fragment contains the 5'-flanking region, the selective marker gene is preferably under the control of the endogenous promoter A.niger, and 3'-flanking region, and they all have the same direction and the same orientation. DNA end structures clone into a vector that can replicate in E. coli.

Inactivating the split design appropriate restrictable to remove vector sequences from E. coli, and inactivating the fragment allocate standard methods (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). And finally, Aspergillus niger transform inactivating fragment using the method described in the literature is the temperature, for example, the method described by Kusters-van Someren et al. (1991) Curr. Genet. 20, 293-299. Transformed cells are selected by sowing a mixture for transformation to plates with agar, which is selective for the growth of strains of Aspergillus niger expressing the marker gene. After cleaning the transformed strains of Aspergillus by sowing the replicas of a number of representative strains analyzed by the method of southern blotting using standard techniques (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). For this, genomic DNA of transformed mycelium strains isolated and digested with suitable restrictase. Restriction fragments separated using electrophoresis on agarose gel, subjected to blotting on nitrocellulose membranes and probe labeled fragment of the marker gene. Hybridization and washing is carried out in harsh conditions. Suitable are strains that contain the labeled restriction fragments of the desired length. This method can be selected A.niger strains containing the inactivated protease gene.

Example 4

The secretion of proteases by using ion-exchange chromatography

A small amount of protease encoded described herein nucleotide sequence was obtained by constructing expressing plasmids containing the relevant the NC sequence; transformation of A.niger strain by plasmid; and cultivation of A.niger strain in a suitable medium. After collecting the broth not containing contaminating cells, the desired protease can be purified.

To highlight the protease encoded considered a nucleotide sequence essentially in pure form can be used different strategies. All these strategies are well described in the relevant scientific literature (see, for example, manual Protein Purification Handbook, 18-1132-29, Edition AA, Amersham Pharmacia Biotech, Uppsala, Sweden). This guide also describes the procedure is suitable for the purification of proteases from complex mixtures. It is important that the appropriate analysis is available and selective in relation to the properties of the enzymes. For proteases commonly used chromogenic synthetic peptide substrate described in example 1. Such peptide substrates can be selective for endoprotease, carboxypeptidase, aminopeptidase or omegareplica. In example 11 described selectivity for specific tripeptidylpeptidase. Protease with the desired specificity can be selected by selecting the corresponding amino acid residues in the relevant synthetic peptide.

First you need to determine whether secreted this protease in the environment depending on the expression system chosen for the Pro is ucirvine protease, it can secretariats of cells, or contained in the cell. If the protease is secreted into the fermentation medium, producing cells or fragments of these cells should be removed by centrifugation or filtration, and the obtained transparent or clarified environment can be used as source material for further treatment. If this protease is not secreted, then producing cells need to be disruptive to highlight protease. In such cases, the collected cell mass is finely chopped with the use of abrasive, to grind in a ball mill, and subjected to ultrasonic treatment and processing on the press of the French or in the homogenizer of Mentana-Galina, and then filtered or centrifuged. If this prosthesis is hydrophobic or membrane-associated, before stage filtration or centrifugation may be necessary to add a non-ionic detergent to solubilize protease. To obtain unknown proteases, essentially in its pure form, after the stage of clarification can be done three phase purification strategy. All or some of these three phases may be necessary to add detergent.

In the first phase, or in phase immobilization, the desired protease exhale, partially purified and end tryout. In a subsequent intermediate stage of purification a large part of the total impurities are removed, and in the final stage of purification to remove trace amounts of remaining impurities larger closely related substances, and the enzyme is dissolved in the appropriate buffer. The specialist may, depending on the nature and physical properties of the investigational protease to optimize these three phases, using slightly modified versions of various binding protein materials and changing to a certain extent the conditions. However, in all cases it is necessary to conduct selective analytical test because it allows continuous monitoring of the increasing activity of the purified proteolytic enzyme. Analytical tests that are suitable for these purposes include the use of chromogenic peptide substrates mentioned above.

In the first immobilsarda the cleanup phase, it is preferable to use a strong anionic ion-exchange resin type for clarification and desalted enzyme-containing environment. In order to guarantee the desired binding proteolytic activity with the resin being tested at three or four different values of pH and resin under conditions of low conductivity. In these tests the resin always balance the buffer with the same pH and conductivity as in the enzyme-containing create environment is applied to the column under conditions of pH, which, as has been shown, allow for adequate binding of the protease with the resin, so that none of the desired enzymatic activity was not detected in on-going environment. Then the desired enzymatic activity elute from the ion exchange resin using a continuous salt gradient, where the gradient elution starting with balanced resin buffer and finish the same buffer, to which was added 1 mol/liter NaCl. In accordance with this analysis erwerbende fractions containing the desired activity, collect, and then cook for an additional stage of treatment. This additional stage of treatment depends on the purity of the desired enzyme in the collected fractions: if it is almost clear, it is necessary to conduct additional adequate stage of gel filtration; and if it is not clean enough, it is necessary to chromatography on hydrophobic resin, and then the stage of gel filtration.

Chromatography on hydrophobic resin was carried out first by increasing the salt content in the collected fractions obtained from ion exchange resin, up to 4 mol/liter NaCl, followed by removing any precipitate formed. If you receive the clarified fraction does not contain the desired activity, then this activity is obviously present in the sediment and may not be allocated the Lena in essentially pure form. If this analysis of the obtained transparent faction continues to possess the desired activity, then this liquid is applied on phenyltetrazol resin (Pharmacia), equilibrated in buffer with high salt concentration, having the same pH and conductivity. If the fraction with the desired enzymatic activity associated with phenyltetrazol resin, this fraction elute continuous gradient with decreasing salt content, and then washing, not containing salt, and if necessary, chaotropes agent. As described earlier, fractions, obtained with a gradient elution of fractions and active in the analysis, collected and in the end was subjected to gel filtration. If the fraction with the desired enzymatic activity is not associated with phenyltetrazol resin, in this case, there are a lot of impurities, and therefore, to obtain the desired proteolytic activity present in the void volume of the column requires only additional stages of ultrafiltration in order to obtain activity in a more concentrated form, which is then applied to a column for gel filtration. Column gel chromatography allows not only to remove trace impurities, but also to introduce the enzyme into the buffer, which is required for later use.

Although this method is mainly suitable for separation and cleaning the protease of the present invention, however, in example 4, described in more specific allocation method. This example describes the selection of the protease of Aspergillus using immobilized bacitracin, peptide-antibiotic which is known to selectively interacts with the proteases of different types.

Example 5

The secretion of proteases by using affinity chromatography

An alternative method of purification of small amounts of protease is affinity chromatography. To obtain protease in purified form 100 ml culture was grown in a well-ventilated shaker flask. After centrifugation to remove any insoluble substances supernatant was applied to a 40 ml column with bacitracin-separate, balanced 0.05 mol/liter sodium acetate, pH 5.0. Protease associated with the column, was suirable acetate buffer, to which was added 1 mol/liter NaCl and 10% (V/V) isopropanol (J. Appl. Biochem. 1983, pp.420-428). Active fractions were collected, were dialyzed against distilled water and was applied to a 20-ml column with bacitracin-separate, then again balanced by acetate buffer. As before, the elution was performed using acetate buffer, to which were added NaCl and isopropanol. Active fractions, i.e. fractions exhibiting the desired activity, were collected, were dialyzed against 5 millimoles/liter of catatoga buffer, pH 5.0, and then concentrated by ultrafiltration on a membrane Amicon PM-10. To obtain essentially pure protease concentrated liquid was chromatographically on column superdex 75, balanced 0.05 mol/liter nitroacetate buffer, pH 5.0, to which was added 0.5 mol/liter NaCl. Subsequent experiments using purified enzyme by electrophoresis on PAG, can confirm whether its molecular weight with a molecular mass predicted from existing data about the sequence. Final confirmation can be obtained by the analysis of incomplete N-terminal amino acid sequence.

Example 6

Properties of a new cysteine protease originating from A.niger

In this example, the gene of Aspergillus No. 28 cloned and sverkhekspressiya in A.niger, as described above. The obtained enzyme was purified in accordance with the procedures described in example 4 and used to destroy the trypsin-inhibitory activity of soybean under different conditions. As reference materials used papain and bromelain. Bromelain was obtained from the company Sigma, and papain was obtained from the company DSM Food Specialties Business Unit Beverage Ingredients, PO Box 1, 2600 MA Delft, the Netherlands.

Inhibition of trypsin was measured by the method described Kakade M.L., Rackis, J.J., McGhee, J.E. & Puski, G. (1974): J. Cereal Chemistry 51:376-382. is to measure the activity of trypsin was carried out by cleavage of the substrate N-benzoyl-L-arginine-p-nitroaniline N-benzoyl-L-arginine and p-nitroaniline. Trypsin was purchased from British Drug Houses Ltd and received from bovine pancreas, containing more than 0,54 Anson units per gram of product. Soybean inhibitor Marten was obtained from Sigma.

This trypsin inhibitor pre-incubated at a concentration of 2 mg/ml with the above enzymes of the serine proteases at pH 3 in 50 mm Na-acetate buffer, and the subsequent evaluation of the inhibition of trypsin. The enzymes were added in the ratio of enzyme protein:trypsin inhibitor, 1:100 (mass/mass). As a negative control for enzymes served as albumin. Residual Triesenberg activity was measured after incubation for 3 hours at 37aboutC. the Results are presented in table 2.

Table 2

The effect of various cysteine proteases

the enzymatic inactivation of trypsin inhibitor Marten isolated from soybeans
12345
The tested enzymeThe residual activity of TI (%)The residual activity of TI after treatment with pepsinThe residual activity of TI after heat treatment at 75°The residual activity of TI after heat treatment at 90°
Papain25 557895
Bromelain30628699
A.niger26262835
Albumin (control)100100100100
TI: the activity of trypsin inhibitor

The experiments were repeated in the presence of pepsin in the process of pre-incubation cysteine proteases with trypsin inhibitor. Pepsin was added at a final concentration of 1.3 mg/ml the Results are presented in column 3.

Other series of experiments was carried out to evaluate thermal stability. Cysteine protease incubated at 75 and 90°C for 5 minutes and then added enzymes for pre-incubation with inhibitors of trypsin. The results are presented in columns 4 and 5.

These results clearly demonstrated excellent activity new cysteine proteases of Aspergillus niger in comparison with the already known cysteine-proteases used for inactivation of trypsin inhibitors in animal feed.

Example 7

Ectopeptidases stimulating the maturation of the cheese and the formation of cheese taste

Aminopeptidase, encoded by the genes No. 20 and 54 (see table 1), were sverkhekspressiya in A.nige methods described above. Purification of these enzymes was carried out in accordance with the procedures described in example 4. The activity of samples of purified enzyme was determined at pH 7.2 in aqueous phosphate buffer (50 mm)containing para-nitroaniline derived series of hydrophobic amino acids (3 mm) as substrate. The transformation of the substrate aminopeptidase was evaluated by monitoring changes in optical density at 400 nm in the transformation of the substrate, the control solution was used, containing no enzyme. Activity (A) was calculated as the change in OD per minute and expressed, for example, as units Phe-AR, Leu-AR or Val-AR, depending on the substrate. Normal milk for cheese production was inoculable starter culture Delvo-tecTMDX 31 (DSM Food Specialities Delft, The Netherlands) to get cheese like Gouda, and coagulation was performed using medium doses of coagulant (50 IMCU per liter syroprigodnogo milk). In addition, two experimental cheeses were added 25 Phe-units each ectoprocta, whereas the control did not contain any ectoprocta. The parameters used in obtaining both cheeses, consistent with the parameters used in the production of semi-hard cheeses. In the formation of taste and aroma between experimental cheeses and control cheese was observed differences, and the time, experimental cheeses have gained most of its organoleptic properties of three (3) weeks, whereas the control cheese was purchased similar properties in six (6) weeks. It was shown that the experimental cheeses level of free amino acids in three weeks was twice as high, and six weeks of ripening of the cheese he was again comparable. Analysis of amino acids was performed by the Picotag method, Waters (Milford, MA, USA).

These data suggest that the product was ready for sale three weeks early and by as he was not inferior to the quality of normal cheese. Organoleptics properties of the experimental cheeses differed from the organoleptic properties of the control cheese to the fact that in contrast to the control cheese in experimental cheeses in the presence of amino peptidases were gone mild, slightly bitter taste. It was found that the texture of these cheeses was also somewhat more delicate.

Example 8

The new specificity of the protease encoded by the genome 55

As explained above, some proteins can be resistant to enzymatic hydrolysis, due to specific amino acid composition or the presence of specific tertiary structures. In such cases, the number of peptides that are present in these resistant to the protease proteins and that mo the ut to be solubilisation, can be dramatically increased with the use of proteases with new specificnosti.

Beta-casein is a protein that has a very limited tertiary structure, but an exceptionally high level prolinnova residues. Many proteases can't split polysterene sequence, and therefore the hydrolysis of beta-casein known proteases gives hydrolyzate, is relatively enriched in large resistant to the protease peptides. These resistant peptides may be responsible for a number of undesirable properties of the hydrolysate. For example, it is well known that these larger peptides have a relatively strong effect from the point of view of allergenicity and have a bitter taste. Moreover, these peptides are resistant to further decomposition into free amino acids, and therefore, in some processes, the presence of these large-resistant protease peptides equivalent to a yield loss. Therefore, the availability and use of proteases that can cleave resistant to the protease part of the protein, will contribute significantly to technological progress and economic benefit.

Beta-casein is one of the major casein fractions of cow's milk. This protein has been well characterized in terms of amino acid sequence and the C is commercially available in almost pure form. Essentially, beta-casein is an excellent test substrate used to study the relationship between the sites of cleavage by the enzyme and the length of various peptides formed during enzymatic hydrolysis.

This example illustrates that despite the wide range of cleft endoprotease subtilisin, adding a highly specific enzyme, such as polyangiitis encoded by the genome of 55 (see table 1), has a huge impact on the size of the formed fragments of beta-casein.

Beta-casein of cow's milk (liofilizirovannogo essentially not containing salt dry milk), contains at least 90% beta-casein was purchased from Sigma. Subtilisin isolated from B.licheniformis (Delvolase ®, the 560,000 DU per gram), was purchased from DSM Food Specialities (Seclin, France). Proline-specific endopeptidase encoded by the genome of 55, was sverkhekspressiya in A.niger and purified using the procedures described in example 4.

Powder beta-casein was dissolved at a concentration of 10 wt.% together with 0.1 wt.% powder DelvolaseTMin 0.1 mol/liter phosphate buffer pH 7.0. After incubation for 24 hours at 45°C in a water bath with shaking, the reaction was stopped by heating the solution for 15 minutes at 90°C. one half of the solution (1 ml containing 100 milligrams of beta to the Zein) was added 100 microliters Proline-specific protease and the reaction continued for another 24 hours at 45° C. After heat shock at 90°With samples of beta-casein treated DelvolaseTMand DelvolaseTM+ Proline-specific protease, and analyzed using LC/MS equipment to study the exact distribution of the peptides in the two samples.

LC/MS analysis

Characterization of enzymatic hydrolysates of protein produced using a mixture of enzymes of the present invention, was performed using HPLC with the use of a mass spectrometer with an ion trap (ThermoquestTM, Breda, The Netherlands)connected to the pump R (ThermoquestTM, Breda, The Netherlands). The resulting peptides were separated on a column (PEPMAP C18 300A (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands), elwira gradient of 0.1% formic acid in water Milli Q (Millipore, Bedford, MA, USA; solution A) and 0.1% formic acid in acetonitrile (solution B). This gradient elution was started with 100% solution A, increasing to 70% solution In 45 minutes, and maintained at this rate for another 5 minutes. Used the volume of injection was 50 microliters, the flow rate was 50 microliters per minute and the column temperature was maintained at 30°C. the protein Concentration of injected sample was approximately 50 micrograms/milliliter.

Detailed information about the individual peptides was obtained using were elaborated to the and depending on the scan MS/MS, which is a characteristic algorithm for mass spectrometer with an ion trap. After the complete scanning analysis was performed scanning analysis by scaling to determine the charge of the most powerful ion across the scanned mass. Subsequent MS/MS analysis later Jonah gave incomplete information about the peptide sequence, which can be used for database searches using application SEQUEST from Xcalibur Bioworks (ThermoquestTM, Breda, The Netherlands). Used databases were taken from the data Bank OWL.fasta available in NCBI (national center for biotechnology information) and containing the proteins of interest for the application.

With the use of this technique as a method of scanning as peptides that are suitable for subsequent analysis by MS-sequencing, were considered only peptides with a mass in the range of about 400-2000 Yes.

Angiotensin (M=1295,6) used for setting the optimal sensitivity in MS for optimal fragmentation in MS/MS, in the implementation of continuous infusion of 60 µg/ml, which was led mainly to obtain double and treasuretrove ions in MS, and optimal energy collisions of approximately 35% in MS/MS.

In the sample, split only Delvolase, L/MS/MS analysis allowed us to identify 40 peptides covering different parts of the molecule beta-casein. Together, these peptides constitute 79% of the total sequence of the beta-casein. It was found that peptides with a length in the range from 2 to 23 amino acid residues have different retention time on C18 column. It was found that in General <15% of the peptides have a size of less than 6 amino acids. In the sample, split DelvolaseTMand the Proline-specific protease, also formed a large number of peptides identified in the beta-casein. Together, these peptides covered >50% of the entire sequence of the protein beta-casein. In the specified sample distribution of peptides in size was fairly homogeneous, since these peptides had a length of only in the range of 2 to 6 residues. The results showed that the hydrolysate obtained with the use of a Proline-specific protease, contained a large fraction of peptides having 2 to 6 amino acids, indicating a clearly positive effect of joint incubation with endoproteases with unusual specificity. From these experiments it is evident that endoprotease encoded by the genome of 55, is endoprotease, which cleaves the peptide chain from carboxylic prolinnova balance.

Example 9

Selective release of specific amino acids to stimulate formation of the Oia taste

Free amino acids such as leucine and phenylalanine are not only involved in Maillard reactions, but also are precursors forming the desired odors in fermentation of various food products. For stimulation of the formation of such odors in the fermentation process of food or in the process of heating, frying or baking food may be preferable to introduce these products are hydrolyzed protein, which contains relatively high levels of these specific amino acids in free form. In this example, the authors describe getting yeast extracts selectively enriched in leucine and phenylalanine. This enrichment is achieved by combining endoprotease, with a preference for cleavage of a selected number of amino acid residues, with ectoprocta, which has a preference to release a similar number of amino acid residues. This preference is specified endoprotease must match the preference used ectoprocta. For example, the authors found that amino peptidases encoded by the genes of 20 and 54 (see table 1), have a certain preference to the release latinovich and phenylalanine residues, which coincides with the preference thermolysin in his gidrolizuet action. Carboxypeptidase, tiruemye genes 23 and 24, have a preference for the release of arginine and lysine residues, which coincides with the preference of trypsin in his gidrolizuet action. The carboxypeptidase encoded by gene 5, has a highly unusual preference to the release of glycine and can be combined with some endoprotease present in papain. The carboxypeptidase encoded by the genome 51 that can remove the remnants of glutamate, and this ability coincides with the ability of glutamate-specific protease encoded by the genome of 43.

It is known that endoprotease thermolysin (commercially available under the name termbase) s supplied by the company Daiwa Kasei K.K. (Osaka, Japan), cleaves the peptide bond on the part of aminocore volume of hydrophobic amino acids such as Leu and Phe. To release the thus treated amino acids from peptides that have been created by the authors were used amino peptidases encoded by genes No. 20 and 54 (see table 1). These genes were sverkhekspressiya in A.niger previously described methods, and the purification of these enzymes was carried out in accordance with the procedures described in example 4.

Obviously, for the release of leucine and phenylalanine and, to the extent possible, without concomitant release of unwanted amino acids, using this combination of enzymes, obviously, you need rules is but to choose used in the process of enzymatic hydrolysis. Also present in the yeast endogenous (and probably non-specific) protease, must be inactivated. After a series of test inkubirovanii a Protocol was developed, which uses these two new enzyme and which unexpectedly found selective and efficient release of leucine and phenylalanine of yeast proteins.

For inactivation of yeast endogenous proteases yeast suspension was kept for 5 minutes at 95°C. Then the suspension was rapidly cooled to the desired temperature and the pH was brought to 7.0 using 4n NaOH. Yeast, thermolysin and one of aminopeptidase incubated simultaneously under the conditions described below. After heat shock, pH 2000 ml of yeast suspension was brought to 7.0, after which was added 680 milligrams Termez and after stirring was added purified aminopeptidase. This mixture is incubated under stirring at 50°C for 3 hours and centrifuged. To suppress all enzymatic activities, the pH of the supernatant was brought to 4 and were subjected to additional heat treatment for 45 minutes at 95°C. After an additional centrifugation of the supernatant was taken the sample for amino acid analysis. Precipitated or undissolved matter was removed, centrifuged who eat for 15 minutes at 3500 rpm in megacentre Hereaus Megafuge 2.0R. The supernatant was removed and frozen at -20°C. Immediately after thawing, samples of the supernatant was analyzed for amino acid content by the Picotag method, Waters (Milford, MA, USA).

In these amino acid analysis values for Trp and Cys were lowered, and the values for Asp and Asn summed to obtain a single value. In accordance with the data in the resulting hydrolysate ratio of alanine to Latino (21,3:11,7) was 1:0.5 in. Commercially available yeast hydrolysates usually had a ratio of alanine to Latino 1:0,3.

In the second experiment were obtained yeast extract, which was enriched in free glutamate. To achieve this were used endoprotease (encoded by gene no. 43 according to table 1), with a preference for cleavage at the C-end glutamate residues, and carboxypeptidase (encoded by gene no. 51 according to table 1), capable of removing processed it glutamate residues. Endoprotease encoded by the genome 43, and carboxypeptidase encoded gene 51 (see table 1), were sverkhekspressiya in A.niger methods described previously. Purification of these enzymes was carried out according to the procedures described in example 4.

The main role of free glutamate in a number of processes of formation of odors is well described, and it is known that MSG, the sodium salt of glutamic acid, is the single most important component, sposobstvuyuschim increasing taste.

In this example, the pH of 200 ml of yeast suspension subjected to thermal shock, brought to 8.0, and then added purified enzyme product encoded by the genome 43, and the mixture is incubated for 4 hours at 50°C. Then the pH was lowered to 5.0 and the suspension was centrifuged. To 100 milliliters of the supernatant was added to the purified gene product of a gene 51. Incubation with this carboxypeptidase was carried out for 30 minutes at 50°and at a constant pH correction. After termination of the incubation of the enzyme by heat treatment for 5 minutes at 95°the resulting material was centrifuged (see above) and took a sample for amino acid analysis.

In accordance with the data obtained for the amino acids in the hydrolysate produced, the ratio of alanine to glutamate (30,0:48,7) was 1:1,6. Commercially available yeast hydrolysates usually had a ratio of alanine to glutamate 1:1.

Example 10

Organoleptic evaluation of yeast hydrolysates enriched in specific amino acids

To confirm that the protein hydrolysate of the present invention, enriched in specific amino acids, can generate peculiar smells, has conducted a number of experiments using yeast hydrolysates described in the previous example. For this purpose, received larger fragments atypicalities and liofilizovane. Properties of the obtained powders were compared with the properties of commercially available yeast extract (Gistex LS supplied by the company DSM Food Specialties, Delft, The Netherlands) in a standardized mixture under several reaction conditions. Standardized mixture contained one of the hydrolyzate mixture of grounds and water.

The mixture of bases contained 22 grams of powder (Maxarome Plus Powder (specialized yeast extract with a high content of natural nucleotides, also supplied by the company DSM Food Specialties), 29,2 grams of glucose, 9 grams of fat REFEL-F (hydrogenated soybean oil, supplied by the firm Barentz, Hoofddorp, The Netherlands) and 0.2 grams of staurolite calcium (emulsifier supplied by the company Abitec, Northampton, UK), thoroughly mixed in a mortar.

All standardized mixture contained 5 grams of powdered yeast hydrolysate (i.e. material, enriched or leucine, or glutamate, or commercially available yeast extract), 3 grams of a mixture of bases and 3 grams of water. After thorough mixing of these three suspensions were subjected to heat treatment in different modes, that is either maintained for 65 minutes at 90-95°in the reaction vessel (liquid reaction), or dried under a pressure of 20 mbar at 120°in a vacuum furnace (reaction vacuum frying), or heated in the open Rea is operating the vessel at 120° C for 10 minutes after evaporation of all the water (reaction frying).

After heat treatment, all three products were evaluated on a color ranging from dark brown to almost black. In case of reaction of the vacuum frying was used only faintly colored upper layers. The taste evaluation of the heated product was performed by crushing blackened layers with obtaining fine powders and dissolution of these powders to a concentration of 2% (wt./wt.) in water containing 0,6% (wt./wt.) NaCl. Tasting observations are presented in table 3.

Table 3
ControlLeucineGlutamate
LiquidBroth, easy roastingIced tea, floral, yeast tasteTaste of strong broth, meat flavor, yeast taste
Vacuum fryingThe burning, French friesAstringency, flavor beans, yeast tasteThe burning, broth, yeast taste
RoastingStrong frying, soup, mindsWeak roasting, floral scent, mindsRoasting taste of strong broth, a more pronounced taste minds

Example 11

<> Non-allergenic protein hydrolysates whey formed by tripeptidylpeptidase

Dipeptidylpeptidase, encoded by the genes of 19 and 55, and tripeptidylpeptidase, encoded by the genes 4, 9, 10, 12, 26, 35, 46 and 50 (see table 1), were overproduction, as described above, and they can be purified by the methods described in example 4. After cleaning the optimum pH and stability of each individual enzyme may be established by any available methods known in the art. In addition, the specificity of each enzyme can be determined by methods described in example 1. The selectivity of tripeptidylpeptidase illustrated in the following experiment.

The enzyme encoded by the genome of 12, was overproductive in the cell host Aspergillus niger and purified in accordance with the procedures described in example 4. Thus obtained enzyme were incubated at pH 5 and 50°With various synthetic chromogenic substrates, that is, Ala-Ala-Phe-pNA and Ala-Phe-pNA (both supplied by the company Bachem, Switserland). Incubation with the substrate Ala-Ala-Phe-pNA resulted in a significant increase in optical density at 410 nm, whereas incubation with Ala-Phe-pNA did not give such effect. This observation clearly demonstrated that tripeptidylpeptidase otscheplaut tripeptides and do not show aminopeptidase activity, which can be the cause undesired increase in the number of free amino acids.

In addition, the enzyme encoded by the genome 12, exhibits favorable properties of enzyme stability, as demonstrated in the following experiment. The four samples of the enzyme were incubated at pH 5 for one hour at 0, 40, 50 and 60°C, respectively. Then, each sample of enzyme was incubated with the above-mentioned substrate Ala-Ala-Phe-pNA in citrate buffer at pH 5, and the residual activity in each sample was determined by measuring the increase in optical density at 410 nm. When 0°With sample showed 100%activity at 40°With sample showed 96%residual activity at 50°With sample showed 92%residual activity, and during the 60°With sample showed 88%residual activity.

In the usual way to obtain the hydrolysate with high content of tripeptides whey protein (WPC 75) can be dissolved/suspended in a concentration of 100 grams of protein per liter in an aqueous medium having a pH of 8.5. The first enzyme incubated with subtilisin, endoproteases wide range (Delvolase®, the 560,000 DU per gram, from DSM). After pre-splitting whey this enzyme, taken in a 0.5% concentration of the enzyme per gram of protein for 2 hours at 60°the mixture was subjected to heat treatment to inactivate used endoprotease. Then the temperature was brought to 50°, zabavljanjematchmaking and the entire mixture is incubated until the desired level of tripeptides. The next stage of processing thus obtained hydrolyzate will depend on the goals of the particular application, but they may also include a microfiltration or centrifugation, followed by evaporation and spray drying.

1. Selected polynucleotide, hybridities in conditions of high stringency with polynucleotides coding for tripeptidylpeptidase and having a sequence selected from the group comprising SEQ ID NO:12, 10, 9, 26, 35, 50, 69, 67, 66, 83, 92 and 107.

2. Selected polynucleotide according to claim 1, obtained from filamentous fungi.

3. Selected polynucleotide according to claim 2, obtained from Aspergillus niger.

3. Selected polynucleotide encoding tripeptidylpeptidase (EU 3.4.14.9) with the amino acid sequence corresponding to a sequence selected from the group comprising SEQ ID NO:126, 124, 123, 140, 149 and 164.

5. Selected polynucleotide containing the nucleotide sequence corresponding to the sequence that encodes tripeptidylpeptidase (EU 3.4.14.9) and selected from the group comprising SEQ ID NO:12, 10, 9, 26, 35, 50, 69, 67, 66, 83, 92 and 107.

6. Expression vector containing a polynucleotide sequence according to any one of claims 1 to 5.

7. The expression vector according to claim 6, characterized in that the polynucleotide sequence according to any one of claims 1 to 5, functionally linked to regulatory consequently the difficulties, suitable for the expression of the specified polynucleotide sequence in a suitable cell host.

8. The expression vector according to claim 7, characterized in that specified a suitable cell host are filamentous fungi.

9. The selected polypeptide with the activity of tripeptidylpeptidase (EU 3.4.14.9)corresponding to a sequence selected from the group comprising SEQ ID NO:126, 124, 123, 140, 149 and 164, which can be obtained by expression of polynucleotide according to claims 1-5, or vector for p-8 in the corresponding cells of the host.

10. The selected polypeptide according to claim 9, obtained from Aspergillus niger.

11. The method of producing the polypeptide according to any one of claim 9 or 10, providing for transforming a suitable host cell selected polynucleotide according to any one of claims 1 to 5 or a vector according to any one of p-8, cultivation of specified cells under conditions suitable for expression of the indicated polynucleotide, and, optionally, purification of the polypeptide from the specified cell or culture medium.

12. Recombinant cell host containing polynucleotide according to any one of claims 1 to 5 or the vector according to any one of p-8.

13. Recombinant a host cell expressing the polypeptide according to any one of p or 10.

14. Method for the diagnosis of Aspergillus infection in the body, including the stage (a) allocating a biological sample from the specified organism, which, as the site is planned, with Aspergillus,

b) release of nucleic acid from the specified sample,

(C) determine whether the specified selected nucleic acid polynucleotide, gibridizatsii with polynucleotide according to any one of claims 1 to 5.

Priority items:

12.07.2001 EP 01000280.6 - p.1-14 SEQ ID NO:9 and 123;

09.08.2001 EP 01000357.2 - p.1-14 SEQ ID NO:10 and 124;

21.05.2001 EP 01000159.2 - p.1-14 SEQ ID NO:12 and 126;

30.07.2001 EP 01000323.4 - p.1-14 SEQ ID NO:26 and 140;

23.02.2001 EP 01200660.7 - p.1-14 SEQ ID NO:35 and 149;

20.09.2001 EP 01000478.6 - p.1-14 SEQ ID NO:50 and 164.



 

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