Trans-sialydase from trypanosoma congolense

FIELD: medicine.

SUBSTANCE: trans-sialydase enzyme has been recovered from a unicell Trypanosoma congolense. Trans-sialydase is characterised by one of the following amivo acid sequences: SEQ ID NO:2, SEQ ID NO:4 or a sequence being 75% identical with one of said sequences.

EFFECT: extended range of enzymes with trans-sialydase activity.

6 cl, 3 dwg, 1 tbl, 6 ex

 

The subject of invention

The present invention relates to new enzymes that transfer sialic acid from the donor molecule (e.g., oligosaccharides, polisialovoi acids, glycated proteins, glycated peptides, glycated lipids (e.g., gangliosides) and other glycosylated low molecular weight and high molecular weight molecules) on the acceptor molecule (e.g., oligo - and polysaccharides, glycated proteins, glycated peptides, glycated lipids and other glycosylated low molecular weight and high molecular weight molecules) (TRANS-sialidase). These enzymes were isolated from the unicellular organism Trypanosoma congolense.

In addition, the invention relates to functional equivalents of these enzymes; encoding these enzymes and their functional equivalents to the sequences of nucleic acids; expression constructs and vectors containing these sequences; recombinant microorganisms that carry the coding sequence of the nucleic acid according to the invention; methods of recombinant obtain enzymes according to the invention; methods of isolation of enzymes according to the invention of Trypanosoma congolense; methods of enzymatic sililirovanie acceptor molecules using enzymes according to the invention; effectors Proc. of the na-sialidase according to the invention; the application of sequences of nucleic acids, enzymes, effector or products sililirovanie according to the invention for the production of vaccines, medicines, food or food additives; and products obtained according to the invention.

The level of technology

TRANS-sialidase can transfer sialic acid, preferably alpha-2,3-linked sialic acid from the donor molecule to the acceptor molecule, thus again formed alpha-2,3-glycosidic bonds, preferably β-terminal galactose residue.

The term sialic acid understood by all N - and O-derivatives of neuraminic acid (Blix et al., 1957). Narimanova acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-nonoo-pyranose-acid) is an amino sugar with a skeleton of the nine carbon atoms, which is due to the carboxyl group at the second C-atom becomes very acidic RK-figure 2.2 and is therefore negatively charged at physiological conditions. Unsubstituted form is very unstable and does not occur in nature in free form (Schauer, 1982). However, now known for more than 40 natural derivatives of neuraminic acid (Schauer and Kamerling, 1997). The two most frequently occurring sialic acids are N-acetylneuraminic acid (Neu5Ac), the forerunner of all glycoside with the provided sialic acids (Schauer, 1991), and N-glycolylneuraminic acid (Neu5Gc), which arises as a result of hydroxylation of the methyl group of N-acetyl residue CMP-Neu5Ac (Shaw und Schauer, 1988). Hydroxyl group these two sialic acids may be replaced by an acetyl, latinname, sulfate and phosphate residues in various combinations, resulting in a large structural diversity of sialic acids (Schauer, 1991; Schauer and Kamerling, 1997).

The largest portion of the naturally occurring sialic acids exists in bound form as a component of oligosaccharides, polysaccharides, and especially glycoconjugates (Schauer, 1982). But also known policially acid from transgenic microbial production. Sililirovanie the glycoconjugates are found primarily in the outer membrane of cells, but are also important components of serum and mucosal mucus (Traving and Schauer, 1998). Sialic acid protects glycoproteins and cells from attack by proteases and other enzymes and, consequently, the splitting (Reuter et al., 1988). Containing sialic acid mucous membranes of the gastrointestinal tract to form not only protection from digestive enzymes, but also protects the underlying tissue from the penetration of pathogenic bacteria (Keim and Schauer, 1997).

A very important function of sialic acids in molecular and cellular recognition processes. If this is what they mask the receptors and thus prevent the interactions between receptors and ligands (Schauer, 1985; Kelm and Schauer, 1997). For example, sialic acid protects glycoproteins in the serum and erythrocytes from splitting, and phagocytosis, masking underlying galactose residues. When the removal of terminal sialic acids subterminal galactose residues may contact the lectins on hepatocytes or phagocytes, and mouse serum proteins or erythrocytes. The following example is the protection of homologous tissues, as well as many vysokomehanizirovannyh tumors from recognition with the help of the immune system (Pilatte et al., 1993). In case of loss of the protecting layer of sialic acids can occur autoimmune reactions.

Sialic acids also serve as recognition sites for homologous cells and hormones and, therefore, play an important role in cell-cell interaction (Kelm and Schauer, 1997). Inflammation, for example, endothelial cells Express on their surface the selectins, which recognize certain sililirovanie patterns (for example, sialyl-Lewis X) on leukocytes, so that they are associated with endothelial cells and can penetrate into the tissue (Lasky, 1995). In addition, the effect of TRANS-sialidase affects the activation of T cells humoral immune defense (Gao et al., 2001). Sialoadhesin (Siglecs), such as myelin-associated glycoprotein (MAG), also bind with high specificity with cialisyou the governmental glycanase (Kelm et al., 1996; Crocker et al., 1998). Myelin-associated glycoprotein involved in the nervous system, in particular, in myelination and in the regulation of axon growth. Thus, it is not surprising that recently it was found that TRANS-sialidase by transfer of sialic acids are involved in the differentiation of nerve cells and glial cells (Chuenkova et al., 2001). CD-22 is the next binding sialic acid receptor, which is found on lymphocytes and makes possible "dialogue" T - and b-lymphocytes. Family Siglecs is an average of more than 10 molecular-biologically characterized predecessors.

However, sialic acid is important not only when homologous recognition processes, but are also receptors for some bacteria, viruses and toxins. So, for example, is the binding of tetanus toxin with gangliosides of the nervous synapses via sialic acids (Schauer et al., 1995). Specific in respect of sialic acids adhesion through microbial lectins (Sharon and Lis, 1997) often is a decisive stage in infectious diseases, for example, when caused by some strains of E. coli neonatal meningitis or infections of the gastric mucosa of Helicobacter pylori. First of all, the causative agents of influenza a and b are attached via sialic acid to the cells that have become infected (Schauer, 2000).

M is the modification of sialic acids, in particular, O-acetylation, are of great importance in the regulation of molecular and cellular recognition (Schauer, 1991). Thus, the influenza viruses With contact specific 9-O-acetylated sialic acids on the epithelium of the bronchi (Herrler et al., 1985), whereas O-acetylation inhibits the binding of influenza viruses a and b (Higa et al., 1985). But first of all O-acetylation of sialic acids is very important for the morphogenesis and development of various tissues (Varki et al., 1991). In the case of neuroectodermal tumors rises (Hubl et al., 2000; Fahr und Schauer, 2001), and in colon cancer decreases (Cornfield et al., 1999). Sialic acids are essential modulators of the biological behavior of tumors (Schauer, 2000).

Description of figures

Figure 1 shows a comparison of amino acid sequences of TRANS-sialidase TS1 and TS2 according to the invention. Identical amino acids in the two sequences otmani as dark areas. Match (identity) of the two partial sequences is only about 50%.

Figure 2 shows the different types of reactions sialidase, sialyltransferase and TRANS-sialidase.

Figure 3 shows a comparison of the amino acid sequence of sialidase of Trypanosoma rangeli (T. r. S), TRANS-sialidase Trypanosoma cruzi (T. cr. TS) and TRANS-sialidase Trypanosoma brucei brucei (T. b. br. TS) with partial sequences of both Tran is-sialidase of Trypanosoma congolense (T. con. TS1 and T. con. TS2) according to the invention. Amino acids in all sequences are identical, depicted in white on a dark gray background. Amino acids that are identical in at least 4 of the 5 sequences, printed in black on a dark gray background, whereas amino acids that are identical in at least 3 of the 5 sequences depicted in light gray color.

The invention

The objective of the invention was to develop a new means by which to influence adjustable sialic acid biological or pathobiological processes.

The above problem is an unexpected way to solve it with the help of new enzymes from the TRANS-sialidase activity and their coding sequences from Trypanosoma congolense.

The first subject of the invention relates to polynucleotides that encode proteins with TRANS-sialidase activity and can be isolated from Trypanosoma congolense, and these proteins preferentially catalyze the transfer of sialic acid from the donor molecule to the acceptor molecule.

Preferred polynucleotide include at least one nucleic acid sequence in accordance with SEQ ID NO:1 or 3 or represent fragments that include at least 15 of linked nucleotide residues. Complement the MD of the invention are also complementary to them polynucleotide and fragments; and the nucleotide sequence produced from these polynucleotides due to the degeneracy of the genetic code.

The next subject of the invention relates to oligonucleotides that hybridize with polynucleotides according to the invention, in particular, under strict conditions.

The subject invention are also polynucleotides, which hybridize with the oligonucleotide in accordance with the above definition, in particular, under stringent conditions and encode a gene product from microorganisms of the genus Trypanosoma.

The subject invention also include polypeptides that are encoded by polynucleotides, which comprises a sequence of nucleic acid according to the definition above; or which detect amino acid sequence that includes at least 10 related amino acids according to SEQ ID NO:2 or 4; and their functional equivalents, which have TRANS-sialidase activity.

The subject of the invention are, in particular, TRANS-sialidase or their functional equivalents to the TRANS-sialidase activity, characterized by the following partial amino acid sequences:

TDTVAKYSTDGGRTWKREVIIPNGR (position 1-25 according to SEQ ID NO:2)

FRIPSLVEIDGVLIATFDTRYLRASDSSLI (position 1-30 according to SEQ ID NO:4)

Preferred Tran is sialidase 1 (TS1) is at least one of the following characteristics:

The nucleotide sequenceSEQ ID NO:1
Amino acid sequenceSEQ ID NO:2
Temperature optimum30-40°C
The optimum pHpH 6,5-8,5
Isoelectric pointpH 4-5
The molecular mass of the native electrophoresis400-600 kDa
Molecular weight in pampering electrophoresis LTO-PAG90 kDa

Another preferred TRANS-sialidase 2 (TS2) is at least one of the following characteristics:

The nucleotide sequenceSEQ ID NO: 3
Amino acid sequenceSEQ ID NO: 4
Temperature optimum30-40°C
The optimum pHpH 6,5-8,5
Isoelectric point pH 5-6
The molecular mass of the native electrophoresis120-180 kDa
Molecular weight in pampering electrophoresis LTO-PAG90 kDa

The above polynucleotide and polypeptides according to the invention, in particular encoding nucleic acid sequences and amino acid sequences are from an organism Trypanosoma congolense. However, they are also available in synthetic, in particular chemical, biochemical, enzymatic, geotechnologies and transgenic methods.

In addition, the subject invention are functional equivalents of TRANS-sialidase of the present invention.

In addition, the subject invention are expression cassettes comprising a nucleic acid sequence in accordance with the above definition in functional connection with at least one regulatory nucleic acid sequence. In addition, the invention also includes recombinant vectors containing at least one of these expression cassettes.

In addition, the subject invention are prokaryotic and eukaryotic hosts transformed with at least one vector according the above definition.

In addition, the invention relates to the use of the expression cassette, vector or host in accordance with the definition above for recombinant obtain protein with TRANS-sialidase activity.

The subject of the invention is also a method for enzymatic sililirovanie acceptor molecule, wherein the acceptor molecule is incubated with containing residues of sialic acid donor in the presence of TRANS-sialidase in accordance with the above definition and allocate sililirovany acceptor.

Such methods differ in at least one additional property of the following properties:

a) the donor is selected from related oligonucleotides, polysaccharides, policealnymi acids, glycoproteins and glycolipids of sialic acids, such as, in particular, lactoferrin, glycosylated proteins of milk and casein and their fragments;

b) the acceptor is selected from the containing β-galactose polymers, such as β-galactooligosaccharide, lactic, lactobionic acid, methyl-β-lactose, acetyllactosamine, galactopyranoside, TRANS-galactooligosaccharide, Polygalaceae and other glycoconjugates associated with the end of the β(1-3)β(1-4)-galactose; or galactose.

Another aspect of the invention relates to the use of TRANS-sialidase according to the invention, the coding it consistently is ti nucleic acid or received product sililirovanie according to the invention for obtaining a medicinal product, food, food additives or food component for the prevention or treatment of controlled sialic acid parasitic, bacterial or viral infections, for treatment of neoplastic disease; for the treatment of diseases that are associated with the development of tissue; for the treatment of diseases of the immune system; for the treatment of autoimmune reactions; for the treatment of diseases with impaired communication cells; and/or for the treatment of inflammation.

In particular, the subject invention is the use of TRANS-sialidase according to the invention in accordance with the above definition for the development of vaccines against trypanosomiasis or for the development of enzyme inhibitors for the treatment or prevention caused by trypanosome infections.

In addition, the invention relates to the use of TRANS-sialidase, coding its nucleic acid sequence or received in accordance with the invention sililirovany products to obtain medicines, dietary supplements or food product to protect homologous cells or tissues or glycoproteins from enzymatic action.

In addition, the subject invention is the use of TRANS-sialidase, coding its nucleic acid sequence or received in accordance with the invention sililirovany cont the mswb to obtain medicines food additive or food product to influence the development and/or morphogenesis of tissues.

In addition, the invention relates to the effectors TRANS-sialidase activity, selected from

(a) polypeptides of ligands that interact with TRANS-sialidase in accordance with the above definition;

b) low molecular weight effectors that modulate the biological activity of TRANS-sialidase in accordance with the above definition; and

(C) antisense nucleic acids sequences relative to the nucleic acid sequence in accordance with the above definition.

In addition, the invention relates to the use of such effector to obtain medicines, dietary supplements or food product for the treatment or prevention of associated with TRANS-sialidase activity of the disease.

In addition, the subject invention is a method of separation of the enzyme from the TRANS-sialidase activity, in which

a) Trypanosoma congolense cultivate in the environment and

b) the desired product emit ion exchange chromatography using a gradient of salt, if necessary with subsequent isoelectric focusing, gel filtration, affinity chromatography and/or precipitation of the protein.

Finally, the invention relates to a pharmacist who ical or gene therapy tool, containing pharmaceutical or gene therapy carrier at least one effector in accordance with the above definition.

Detailed description of the invention

i) the Value of invention

The value of the invention arises from the possible as a result of implementation of the invention influence on controlled sialic acid mechanisms of parasitic, bacterial and viral infections, effects on cellular communication and immune system and changes in the mechanisms of regulation and development of the tissues of humans and animals, as well as tumors. This aim is achieved by transfer of sialic acids on biologically relevant glycostructures (glikana derived glycans and glycoconjugates) using this TRANS-sialidase.

From the transfer of sialic acids on the selected patterns-media arise, for example, products for the changes of inflammatory reactions, changes of cell interactions in the human body and the animal protection homologous tissues from attack its own immune system (autoimmunity), "rebirth" of cancer cells in the patient's body, whereby homologous immune system again fights with them (cancer therapy and prevention of cancer), suppression of penetration of pathogenic bacteria in the human and animal body, prevention and inhibition is of viral infections of the gastric mucosa by Helicobacter pylori, suppression caused by bacteria and viruses meningitis in newborns, preventive and therapeutic effect on the receptors of eukaryotic and prokaryotic organisms, bacteria, viruses and toxins to avoid deployment of their actions in the human and animal body, the inhibition of binding of cholera toxin to the mucous membranes of the digestive tract of humans and animals, the development of vaccines against trypanosomiasis, the development of enzyme inhibitors to suppress (therapy) of Trypanosoma infections, the impact on the molecular and cellular recognition processes in humans and animals, protection of glycoproteins and cells from attack by proteases and other enzymes, among other things, also for protection from cleavage of molecules by enzymes of the digestive tract human and animal, effect on the development of tissues and effects on the morphogenesis of tissues in the body.

TRANS-sialidase according to the invention are characterized by the following DNA and amino acid sequences, and other DNA sequences of the homologues, for example, with more than 60% compliance (identity) on these partial sequences.

ii) the Data sequences of the preferred TRANS-sialidase

(1) Information for the sequence of the enzyme TS1:

Signs of DNA h the primary sequence TS1:

Length: 1491 BP

Type: nucleic acid

Form strand: double strand

Origin: Trypanosoma congolense

The DNA sequence of the enzyme TS1 (SEQ ID NO:1):

Amino acid sequence of the enzyme TS1 (SEQ ID NO:2):

(2) Information for sequence of the enzyme TS2:

The signs of the partial DNA sequence TS1:

Length: 831 BP

Type: nucleic acid

Form strand: double strand

Origin: Trypanosoma congolense

The DNA sequence of the enzyme TS2 (SEQ ID NO:3):

Amino acid sequence of the enzyme TS2 (SEQ ID NO:4):

Partial amino acid sequence of the enzyme TS1 and enzyme have TS2 compliance (identity) only about 50%. Therefore, these partial sequences are characterized uniquely as two different substances (see figure 1).

(iii) a description of the properties of newly discovered enzymes TS1 and TS2

a) Physico-chemical properties of these substances

Table 1
Basic data of both TRANS-sialidase TS1 and TS2
PropertiesTS1 TS2
Temperature optimum30-40°C30-40°C
The optimum pHpH 6,5-8,5pH 6,5-8,5
Isoelectric pointpH 4-5pH 5-6
The molecular mass of the native electrophoresis400-600 kDa120-180 kDa
Molecular weight in pampering electrophoresis LTO-PAG90 kDa90 kDa
The influence of salt1 M KCl and NaCl reduces the activity of both enzymes by 50%, desalination again restores enzymatic activity
The influence of metal ions20 mm CA2+, Mg2+, Mn2+: no effect
4 mm Cu2+, Zn2+, Fe2+, Co2+: low influence
The estimated impact of inhibitors10 mm N-(4-nitrophenyl)Aksenova acid: a small inhibition
10 mm N-acetyl-2,3-didehydro-2-deoxyarabinose to the slot: low influence

b) the Biological properties of these substances

Both stated here substances are two enzymes that transfer sialic acid from the donor molecule to the acceptor molecule.

As a donor in the case of both enzymes, a good donor of sialic acids, which can be transported by these enzymes are glikana, for example, oligosaccharides, polysaccharides, policially acid, glycoproteins and glycolipids associated sialic acid. Among the glycoproteins are, in particular, lactoferrin (from humans, cows, goats, sheep, horses, camels and other animals), glycosylated proteins of milk (from humans, cows, goats, sheep, horses, camels and other animals), and other glycosylated proteins of human, animal and plant, and parts thereof, such as, for example, glycomacropeptide of Caseins these animals. Donors can also be used gangliosides.

Both TRANS-sialidase have a good acceptor specificity regarding galactooligosaccharide, in particular, in relation to beta-galactooligosaccharide, such as, for example, Vivinal GOS company Borculo Domo Ingredients (BDI) and Oligomate 55 company Yakult. In addition, as acceptors may act lactic, lactobionic acid, methyl-β-lactose, acetyllactosamine, galactopyranoside, TRANS-g is lactoridaceae, Polygalaceae and other glycoconjugates with terminal β(1,3)- or β(1-4)-linked galactose. Methylation of galactose residues leads to the reduction of the acceptor function. Methylation glucose residues (e.g., lactose) has a negligible effect on the acceptor function. The monosaccharide galactose also serves as an acceptor, although with less specificity.

The enzyme TS1 detects twice more efficient transfer of sialic acid to the corresponding acceptors than the enzyme TS2. The substrates can be free, i.e. soluble or associated with the cell membrane.

Also known transfer alpha-2,3-linked terminal sialic acids on beta-1,4-linked terminal galactose residues of TRANS-sialidase Trypanosoma cruzi (Schenkman et al., 1991; Vandekerckhove et al., 1992; Scudder et al., 1993) and Trypanosoma brucei (Engstler et al., 1992, 1993, 1995). However, TS1 and TS2 are distinguished on the basis of various DNA and amino acid sequences of these already known enzymes. Thus, TS1 and TS2 are characterized as uniquely new substances (TRANS-sialidase). For added distinction, see next paragraph.

iv) the Distinction between the present invention and other TRANS-sialidase, sialidase and sialyltransferase

The enzyme TRANS-sialidase" was first described in the American form of trypanosomes Trypanosoma cruzi (Schenkman et al., 1991). Little is ozdnee failed to detect this enzyme in the American species Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei (Engstler et al., 1993, Pontes de Carvalho et al., 1993, Engstler et al., 1995). In addition, TRANS-sialidase were detected in the species Endothypanum (parasites that infect sloth) (Medina-Acosta et al., 1994), in Corinebacterium diphtheriae (Mattos-Guaraldi et al., 1998) and in plasma (Tertov et al., 2001). Long before the discovery of the TRANS-sialidase have been known so-called sialidase. They are glycohydrolase that transfer sialic acid from the donor molecule exclusively on water, these sialic acids are also formed during the hydrolysis of oligosaccharides and glycoconjugates.

In addition, certain enzymes can tolerate activated citizensinformation (CMP) of sialic acid to other sugar residues, mainly galactose and N-atsetilgalaktozamin. These enzymes are called sialyltransferase (see figure 2).

Stated here TRANS-sialidase transfer of sialic acid is not exclusively from donor molecules to water, as do just sialidase. However, if a suitable acceptor is absent, stated here TRANS-sialidase hydrolyzing sialic acid as simple sialidase. Stated here TRANS-sialidase not need to be activated sialic acids for their reactions migration, as previously mentioned sialyltransferase. TRANS-sialidase also have broader donor and acceptor specificity than acyltransferase, and so they are especially many-sided applicable. Thus, stated here TRANS-sialidase are preferred for industrial applications than just sialidase and sialyltransferase.

So far known only DNA and amino acid sequence of the TRANS-sialidase Trypanosoma cruzi and Trypanosoma brucei brucei, as well as DNA and amino acid sequence of simple sialidase of Trypanosoma rangeli. Stated here the enzyme TS1 has a matching (identity) relative to the corresponding amino acid sequence of the TRANS-sialidase from Trypanosoma brucei brucei below 60% and the line below the 50% relative to the corresponding partial sequence of Trypanosoma cruzi. Stated here the enzyme TS2 has a matching (identity) relative to the corresponding amino acid sequence of the TRANS-sialidase from Trypanosoma brucei brucei below 50% and the line below the 50% relative to the corresponding partial sequence of Trypanosoma cruzi (see figure 3). Further, it is known that amino acids between the TRANS-sialidase of Trypanosoma and famous sialidase and TRANS-sialidase bacteria and viruses is only 20%-30% (Chuenkova et al., 1999, Montagna et al., 2002).

In the case described here, the enzymes we are talking, therefore, about the new characterized substances (enzymes), match (identity) which otnositel the corresponding DNA and amino acid sequences of other known enzymes with similar function is less than 60%.

v) further explanations in regard to the present invention

(a) Polypeptides and functional equivalents

"Polypeptides" in the context of the invention include a characteristic partial fragments of the amino acid sequences according to the invention as well as amino acid sequences of the enzymes according to the invention and their functional equivalents.

Thus, in accordance with the invention, “functional equivalents” or “homologues” are specifically disclosed new polypeptides or enzymes.

“Functional equivalents” or analogs of the specifically disclosed polypeptides are within the scope of the invention which differ from the polypeptide, which, in addition, possess the desired biological activity in accordance with the above definition (for example, substrate specificity).

Under the “functional equivalents” in accordance with the invention, see, in particular, mutants that at least one of the above-mentioned provisions of the sequence find a different amino acid than specifically named amino acid, but, despite this, have specified here biological activity. “Functional equivalents” includes, therefore, the mutants obtained through one or more new is, one or more substitutions, deletions and/or inversions of amino acids, and the above-mentioned changes can be detected at any position of the sequence, until they lead to a mutant with the profile properties in accordance with the invention. Functional equivalence has, in particular, also in the case, if the picture reactivity between mutant and unmodified polypeptide is qualitatively consistent, ie, for example, become the same substrates with different speeds.

"Functional equivalents" in the above sense are also predecessors of the described polypeptides, and functional derivatives and salts of these polypeptides. The term "salt" is understood as the salts of carboxyl groups and acid additive salts of amino groups of protein molecules according to the invention. Salts of carboxyl groups can be obtained in known per se manner and include inorganic salts, such as, for example, salts of sodium, calcium, ammonium, iron, and zinc, as well as salts with organic bases, such as, for example, amines, such as triethanolamine, arginine, lysine, piperidine, etc. Acid additive salts such as, for example, salts with mineral acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid, also t is Auda subject the inventions.

“Functional derivatives” of polypeptides according to the invention can also be obtained on the functional side groups of amino acids or their N - or C-terminal sides using known methods. Such derivatives include, for example, aliphatic ester groups, carboxylic acid amide groups, carboxylic acids, obtained by transformation with ammonia or with primary or secondary amine; N-ACI-derivatives of free amino groups, obtained by transformation with acyl groups; or-ACI-derivatives of free hydroxyl groups, obtained by transformation with acyl groups.

"Functional equivalents" include, of course, also polypeptides that are available from other organisms, as well as naturally occurring variants. For example, by comparing sequence can be installed in areas homologous regions of sequences and application to the specific objectives of the present invention can be obtained equivalent enzymes.

"Functional equivalents" also include fragments, preferably separate domains or motifs, and sequence of the polypeptides of the present invention, which, for example, find the desired biological function.

"Functional equivalents" are, in addition, fused proteins, which detect one in the above-mentioned polypeptide sequences or produced from it functional equivalents and at least one additional functionally different from her, heterologous sequence in functional N - or C-terminal binding (i.e. without mutual significant functional impairment of parts fused protein). Non-restrictive examples of such heterologous sequences are, for example, signal peptides, enzymes, immunoglobulins, surface antigens, receptors or ligands of receptors.

“Functional equivalents” TRANS-sialidase according to the invention are, in particular, enzymes, amino acid sequence or aminokisloty partial sequence which detect relative to the corresponding amino acid sequence or partial amino acid sequence according to SEQ ID NO:2 or 4 sequence identity (homology sequence at least 60%, particularly at least 70%, for example, 75%, 80%, 85%, 90%, 95%, 98% or 99%, calculated in accordance with the algorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448.

In case of a possible glycosylation protein equivalents according to the invention include proteins of the aforementioned type in deglycosylated or glycosylated form as well as the change of the pattern of glycosylation modified forms.

Homologues of proteins and polypeptides according to the invention can be obtained by mutagenesis, for example, by the spectacle of mutation, lengthening or shortening of the protein. The term “homologue” refers in this context to a variant form of the protein, which acts as an agonist or antagonist activity of a protein.

Homologues of proteins according to the invention can be identified by screening combinatorial banks of mutants, for example, truncated mutants. For example, you can create a mosaic Bank protein variants using combinatorial mutagenesis at the level of nucleic acids, for example, by enzymatic ligating a mixture of synthetic oligonucleotides. There are many methods that can be used to obtain the banks ' potential homologs from one degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automated DNA synthesizer, and the synthetic gene can then be Legerova expressing in a suitable vector. Use of a degenerate set of genes allows for the provision of all sequences in one mixture, which encode the desired set of potential protein sequences. Methods of synthesis of degenerate oligonucleotides is well-known specialist in this field (e.g., Narang, S.A. (1982) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem, 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).

b) Pauline is cleotide

"Polynucleotide" in the context of the invention include a characteristic partial fragments of the sequences of the nucleic acids according to the invention that encode the partial amino acid sequences of the enzymes according to the invention as well as nucleic acid sequences that encode the enzymes and their functional equivalents. Preferably, polynucleotide include more than about 20, in particular more than approximately 30, for example, more than about 45 or more than about 60 residues of nucleic acids.

"Oligonucleotides" include, in particular, a sequence of less than about 60, preferably less than about 45, in particular, less than about 30, or less than about 20 residues of nucleic acids.

Everything mentioned here "nucleic acid sequences" can be obtained in known per se manner by chemical synthesis from the nucleotide elementary units, as, for example, by condensation of fragments of individual overlapping, complementary to the basic units of nucleic acid double helix. Chemical synthesis of oligonucleotides can occur, for example, in a known manner, in accordance with phosphoroamidite method (Voet, Voet, 2nd. Aufgabe, Wiley Press New York, Seiten 896-897). Prisoedinenie the synthetic oligonucleotides and filling in the gaps using fragment maple DNA polymerase and ligation reactions, as well as General methods of cloning are described in Sambrook et al. (1989), Molecular Cloning: A Laboratry Manual, Cold Spring Harbor Laboratory Press.

The subject of the invention are nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA)encoding one of the above polypeptides and their functional equivalents, which, for example, available also with the use of synthetic analogues of nucleotides.

The invention relates both to a selected nucleic acid molecules that encode polypeptides or proteins according to the invention or biologically active segments, and to nucleic acid fragments, which, for example, can be used for use as hybridization probes or primers for the identification or amplification of the coding nucleic acid according to the invention.

In addition, the nucleic acid molecule according to the invention may contain untranslated sequences of the 3'and/or 5'-ends of the coding region of the gene.

"Isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid, and it can also be essentially free of other cellular material or culture medium if it is received in recombina time ways, or may not contain chemical precursors or other chemicals, if it is synthesized chemically.

The nucleic acid molecule according to the invention can be isolated using molecular-biological standard techniques and provided by the present invention, the information sequence. For example, cDNA can be isolated from a suitable cDNA Bank using one of the specifically disclosed the full sequence or segment as a hybridization probe and standard ways hybridization (as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratry Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, the nucleic acid molecule that includes the disclosed sequence, or their segment can be selected using oligonucleotide primers that were made on the basis of this sequence. Amplificatory thus nucleic acid can be cloned into an appropriate vector and characterized by sequence analysis (sequencing) DNA. The oligonucleotides according to the invention can be obtained, in addition, the standard synthesis methods, for example, using an automated DNA synthesizer.

The invention also includes nucleic acid molecule, "complementary the e" described specific nucleotide sequences or their segment.

The nucleotide sequences according to the invention make possible the production of probes and primers that are applicable for identifying and/or cloning of homologous sequences in other cell types and organisms. Such probes or primers include typically the region of the nucleotide sequence, under strict conditions hybridizes at least approximately 12, preferably at least about 25, for example, about 40, 50 or 75 consecutive nucleotides of a semantic chain nucleic acid sequence according to the invention or the corresponding antisense chain.

Additional nucleic acid sequences according to the invention produced from SEQ ID NO:1 and 3, and differ from them by the addition, substitution, insertion or deletion of one or several nucleotides, but encode, in addition, the polypeptides with the desired profile of properties.

In accordance with the invention also includes such nucleic acid sequences, which include the so-called silent mutations or altered in accordance with the use of the codons of a special body-the source or the host body, in comparison with a specific named sequence, as well as naturally occurring variants, such as, for example, splicing variations which you or allelic variants. The subject of the invention are also obtained by conservative nucleotide substitutions of the sequence (i.e. the considered amino acid is replaced by an amino acid of similar charge, size, polarity and/or solubility).

The subject invention are molecules produced by polymorphisms of the specific sequences disclosed nucleic acids. These genetic polymorphisms may exist between individuals within a population on the basis of natural variability. These natural variations are usually the variance of 1-5% in the nucleotide sequence of a gene.

In addition, the invention also includes nucleic acid sequences that hybridize with the above coding sequences or are complementary to them. These polynucleotide can be found when viewing genomic banks or cDNA banks and, if necessary, reproduced with appropriate primers using PCR and then, for example, highlighted with appropriate probes. The next possibility is the transformation of suitable microorganisms polynucleotide or vectors according to the invention, the multiplication of these microorganisms and, therefore, polynucleotides, and their subsequent selection. In addition, polynucleotide according to the invention mouttaki be synthesized by chemical means.

Under the property gibridizatsiya” polynucleotide mean the ability of poly - or oligonucleotide to contact under strict conditions with almost complementary sequence, whereas when these conditions are not formed nonspecific relationship between complementary partners. To do this, these sequences should be complementary to 70-100%, preferably up to 90-100%. The property of complementary sequences to specifically communicate with each other using, for example, in Northern or southern blot or primer binding in PCR or RT-PCR. This is typically done by using oligonucleotides with a length of 30 base pairs.

Under “strict conditions” mean conditions, if, for example, after the southern or Northern blotting DNA or RNA fragments on the membrane hybridize with the probe under specific conditions, i.e. at a temperature of 60-70°C (38-42°C at 50% hybridization solution containing 50% formamide). In addition, these conditions are specific or strict, if immediately after hybridization also conduct specific stage of leaching for the elution of non-specific hybridized DNA or RNA probes. At specific stages of leaching it is usually about twice washing at 20-25°C for 5-10 minutes, 2 × SSC-buffer, which contains 0.1% of LTOs (sodium dodecyl sulphate), and sledushij double leaching buffer of lower ionic strength (e.g., of 0.1 × SSC with 0.1% DMN) at a higher temperature (for example, 64°C) [20 × SSC:3 M NaCl, 0.3 M Na-citrate, pH 7.0]. At the same time bound to each other only remain complementary to a high degree of nucleic acid. The establishment of strict conditions known to the person skilled in the art and described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6.

Another aspect of the invention relates to "antimuslim" nucleic acids. They include a sequence of nucleic acid that is complementary to the sequence coding "sense" nucleic acid. The antisense nucleic acid can be complementary to the entire coding circuit or its segment. In the following embodiment, the antisense nucleic acid molecule is antisense relative to the non-coding region of the coding chain nucleic acid sequence. The term "noncoding region" refers to the segments of the sequence, called the 5'- and 3'-untranslated regions.

Antisense oligonucleotide may have a length of, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides. Antisense nucleic acids of the present invention may be constructed using chemical synthesis and enzymatic ligation reactions using well-known this about the region of the ways. The antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides, which are formed in such a way that they increase the biological stability of the molecules or to increase the physical stability of the duplex, which occurs between the antisense and sense nucleic acid. For example, can be used phosphorothioate derivatives and acidentalmente nucleotides. Examples of modified nucleotides that can be used to produce antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-florouracil, 5-iodouracil, gipoksantin, xanthine, 4-acetylcytosine etc.

Antisense molecules of nucleic acids according to the invention is usually injected into the cell or produced in situ, so that they can gibridizatsiya with the cellular mRNA and/or coding DNA or to contact them, so that expression of the protein is inhibited, for example, by inhibiting transcription and/or translation.

The terms “Express” or “strengthening” or “overexpression” is described in the context of the present invention the formation or increase of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA. This can, for example, to enter in on the such gene, to replace the present gene is another gene, to increase chopinot gene or genes, using a strong promoter or using a gene that codes for a corresponding enzyme with a high activity and can, if necessary, to combine these measures.

The terms “loosen” or “to reduce” describe in the context of the present invention weaken or reduce the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA. This can, for example, to deleteroute gene in the body, to replace the present gene is another gene, lowering chopinot transcript of a gene or genes, using a weak promoter or using a gene that codes for a corresponding enzyme with a low activity, and can, if necessary, to combine these measures.

(C) Expression constructs and vectors:

In addition, the subject invention are expression constructs containing under genetic control of regulatory sequences of the nucleic acid sequence of nucleic acid encoding the polypeptide according to the invention; as well as vectors comprising at least one of these expression constructs. Preferably, such constructs of the invention include 5'-against the course of transcription (left) from testwuide coding sequence, the promoter and 3'downstream transcription (right) termination sequence, and also, if necessary, additional regulatory elements, namely, in each case functionally linked to the coding sequence.

Under the “functional linkage” understand the sequential arrangement of promoter, coding sequence, terminator and, if necessary, additional regulatory elements such that each of the regulatory elements can adequately perform its function in the expression of the coding sequence. Examples of functionally related sequences are the target sequences and enhancers, polyadenylation signals, etc. Additional regulatory elements include breeding markers, signal amplification, the sites of initiation of replication (origin) and other Appropriate regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 195, Academic Press, San Diego, CA (1990).

In addition to the artificial regulatory sequences may be present even natural regulatory sequence before the actual structural gene. Through genetic modification, this natural regulation may be, if necessary, off, and gene expression can be raised or lowered. But gene construct can also be built is more just, i.e. not incertitude additional regulatory signals in front of the structural gene and is not removed natural promoter with its regulation. Instead, the natural regulatory sequence mutate in such a way that regulation no longer takes place and the gene expression is increased or decreased. Nucleic acid sequences can be included in this construct, the gene in the form of one or several copies.

Examples of applicable promoters are: cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac, laclq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6, lambda-PR or lambda-PL promoter, which are used preferably in gram-negative bacteria; as well as gram-positive promoters amy and SPO2, in the yeast promoters ADC1, MF, AC, P-60, CYC1, GAPDH or promoters plants CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, or not the promoter of ubiquitin or phaseolin. Especially preferred is the use of inducible promoters, such as, for example, induced by light and especially induced by temperature promoters, such as PrPl-promoter. In principle can be used all natural promoters with their regulatory sequences. In addition, can also preferably be used synthetic promoters.

The above-mentioned regulatory sequences should make possible targeted expression of the sequence is the first nucleic acid and protein expression. This may mean, for example, depending on the host body, which gene is expressed or sverkhekspressiya only after induction, or that it is expressed and/or sverkhekspressiya immediately.

The regulatory sequences or factors can preferably have a positive impact on the expression and thereby raise or lower it. So, may gain regulatory elements preferably at the level of transcription, by using strong transcription signals such as promoters and/or enhancers. Along with this, it is also possible strengthening broadcast improve, for example, stability of mRNA.

Obtaining the expression cassette is performed by fusion of a suitable promoter with a suitable coding sequence of the nucleic acid, and a terminating signal or polyadenylation signal. This is done using conventional recombination and cloning, such as, for example, described in T. Maniatis, E.F., Fritsch and J. Sambrook, Molecular Cloning: A Laboratry Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989), and T.J. Silhavy, M.L.Berman und L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

Recombinant structure of nucleic acid or gene construct insertyour for expression in approach the statutory body of the host in a host-specific vector, which makes the best possible expression of this gene in the host.

"Vectors" are well known to the person skilled in the art and can be taken, for example, "Cloning Vectors" (Pouwels P.H. et al., Hrsg, Elsevier, Amsterdam-New York-Oxford, 1985). Under the vectors should be understood, in addition to plasmids, all other well-known specialist vectors, such as, for example, phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements (mobile nucleotide sequences found in the genomes of bacterial populations), family, Comedy and linear or circular DNA. These vectors may replicate autonomously in the body-the owner or chromosome.

As examples of expression vectors can be identified:

Conventional fused expression vectors such as pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, for K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ), in which glutathione-S-transferase, maltose E binding protein, or protein a is fused with the recombinant target protein.

Expression vectors that are not fused proteins, such as pTrc (Amann et al., (1989) Gene 69:301-315) and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Kalifornien (1990) 60-89).

A yeast expression vector for expression in yeast S. cerevisiae, such as pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Crporation, San Diego, CA). Vectors and methods construction of vectors which are suitable for use in other fungi such as filamentous fungi, include vectors and methods, which are described in detail in van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Hrsg., S. 1-28, Cambridge University Press: Cambridge.

Baculovirus vectors available for expression of proteins in cultured insect cells (or Sf9 cells)include the pAc series (Smith et al., (1983) Mol. Cell Biol., 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

Expression vectors of plants, such as is described in detail in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binary vectors with selectable markers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197; and Bevan, M.W. (1984) “Binary Agrobacterium vectors for plant transformation”, Nucl. Acids Res. 12:8711-8721.

Expression vectors mammals, such as pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).

Additional suitable expression systems for prokaryotic and eukaryotic cells are described in chapters 16 and 17 of Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratry Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

(d) Recombinant microorganisms:

Using vectors according to the invention can be obtained from recombinant microorganisms which, for example, is converted into at least one vector in the image the structure and can be used to produce polypeptides according to the invention. Preferably, the above-described recombinant constructs according to the invention is introduced into a suitable system is the master and Express. Used preferably well-known specialist conventional methods of cloning and transfection, such as, for example, coprecipitate, the fusion of protoplasts, electroporation, retroviral transfection and the like, in order to make the above-mentioned nucleic acid to be expressed in an appropriate expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Hrsg, Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratry Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In accordance with the invention can be obtained also homologous reconstituted microorganisms. To get this vector, which contains at least one segment of the gene of the invention or the coding sequence, which have been introduced, if necessary, deletion, addition or replacement of one amino acid to change the sequence according to the invention, for example, the functional destruction (“knockout”vector). The input sequence can be, for example, also a homologue from a related microorganism or to be produced from the source, which is mammalian, yeast or insect. An alternative, used d the I the homologous recombination vector can be constructed in such a way, that the endogenous gene is mutated in homologous recombination or otherwise altered but still encodes functional protein (e.g., lying to the left (against the course of transcription) regulatory region can be altered in such a way that by means of this changes the expression of the endogenous protein). This modified segment of the gene according to the invention is in the homologous recombination vector. Construction of suitable vectors for homologous recombination are described, for example, in Thomas, K.R. and Capecchi, M.R. (1987) Cell 51:503.

As organisms hosts suitable in principle all organisms that make possible the expression of the nucleic acids according to the invention, their allelic variants, their functional equivalents or derivatives. Under the organisms owners see, for example, bacteria, fungi, yeast, plant cells or animal cells. Preferred organisms are bacteria such as bacteria of the genera Escherichia, such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells of animals or plants, for example, cells Sf0 or SNO.

The selection of successfully transformed organisms can occur with the use of marker genes, which are also contained in the vector or expression cassette. Examples of such marker is ENES are genes of antibiotic resistance genes and enzymes catalyzes a color reaction, which affects the color of transformed cells. They can also be selected using an automated sorting of cells. Successfully transformed vector microorganisms that carry the corresponding genes of resistance to antibiotics (e.g., G418 or hygromycin), can also be selected with the appropriate antibiotic media or culture media. Marker proteins, which are presented on the cell surface can be used for selection using affinity chromatography.

The combination of organisms hosts and suitable for these organisms vectors, such as plasmids, viruses or phages, for example, a plasmid system with RNA polymerase/promoter, phage 8 or 7 or other temperate phages or transposons and/or additional preferred regulatory sequences, forms a system of expression. For example, the term “expression system” should be understood a combination of mammalian cells, such as cells of Cho, and vectors, such as vector pcDNA3neo that are suitable for mammalian cell.

Most preferably, the gene product can also be expressed in transgenic organisms such as transgenic animals, in particular mice, sheep, or transgenic plants.

(e) Recombinant gaining the polypeptides:

In addition, the subject invention are methods of recombinant obtain a polypeptide according to the invention or functional, biologically active fragments, in which producing the polypeptides of the microorganism is cultivated, if necessary, induce the expression of polypeptides and distinguish them from the culture. Polypeptides can thus be produced in large-scale production, if so desired.

Recombinant microorganisms may be cultured and ermentrout according to known methods. Bacteria can multiply in the TV or LB medium and at a temperature of 20-40°C and pH 6-9. In particular, suitable culturing conditions are described, for example, in T. Maniatis, E.F., Fritsch and J. Sambrook, Molecular Cloning: A Laboratry Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

Then these cells, if the polypeptides are secreted into the culture medium, dissolve and the product is obtained according to known methods for isolating proteins from the lysate. These cells can be dissolved on the choice by high-frequency ultrasound, by high pressure, for example, in French-press, through osmolite by treatment with detergents, lytic enzymes or organic solvents, by using a homogenizer or a combination of several of the named who's methods.

Purification of the polypeptides may be achieved by known chromatographic methods, such as chromatography with molecular sieves (gel filtration), such as chromatography using Q-sepharose, ion-exchange chromatography and hydrophobic chromatography, and other conventional methods, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F.G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin. The same applies to the received precombination polypeptides.

Particularly preferably used for the isolation of recombinant proteins vector systems or oligonucleotides which extend the cDNA specific nucleotide sequence and thereby encode elongated polypeptide or fused proteins, which, for example, serve for easier cleaning. Such suitable modifications are, for example, acting as anchors so-called "tags", such as, for example, known as hexa-his-tag anchor modification or epitopes that can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (N.Y.) Press). These anchors can be used to attach proteins to a solid but is Italy, such as a polymeric matrix, which, for example, can be placed in a chromatographic column, or microtiter plate or other media.

Simultaneously, these anchors may also be used for recognition of proteins. In addition, for the recognition of proteins can also be applied to conventional markers, such as fluorescent dyes, enzyme markers, which after reaction with the substrate to form the detected reaction product, or radioactive markers, individually or in combination with anchors for derivatization of proteins.

f) Purification of the desired products sililirovanie of culture

Obtaining a desired product from a microorganism or from the culture supernatant can be performed using various known in the field of methods. If the desired product is not secreted from the cells, the cells can be collected from the culture by slow centrifugation, the cells can be lysed using standard methods, such as mechanical force or sonication. The remains of the cells removed by centrifugation and the supernatant fraction, which contains soluble proteins, receive for additional purification of the desired compounds. If the product cells secreted, the cells are removed from culture by slow centrifugation and the supernatant fraction SOH is anaut for further purification.

The supernatant fraction from both purification methods can be subjected to chromatography with a suitable resin, in which the desired molecule with a higher selectivity than impurities or deducted chromatographic resin, or passes through it. These phase chromatography can if necessary be repeated, and use the same, or other chromatographic resin. Specialist known for the selection of a suitable chromatographic resin and its effective use for certain be treated molecules. The purified product may be concentrated by filtration or ultrafiltration and stored at the temperature at which the stability of this product is maximum.

The prior art many methods of cleaning. These cleaning methods are described, for example, in Bailey, J.E. &Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by methods current level of technology. They include high performance liquid chromatography (VGH), spectroscopic method, the method with a change in color, thin layer chromatography, NIRS, enzyme test or microbiological tests. These methods of analysis are discussed, for example, in: Patek et al. (1994) Appl. Environ. Environ. 60:133-140; Malakchova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioproces Engineer. 19:67-70. Ullman''s Encyclopedia of Industrial Chemistry (1996) Bd. F27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 and S. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.

The following non-limiting examples describe specific embodiments of the invention.

The examples of the preparation, purification and use stated here TRANS-sialidase

General part:

Conducted within the framework of the present invention the stage of cloning, such as, for example, splitting restrictase, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of cells, growing bacteria, the multiplication of phages and sequence analysis (sequencing) of recombinant DNA were performed as described by Sambrook et al. (1989) in the above location.

Example 1: Isolation of the enzyme from cultures of Trypanosoma congolense

Pro-form Trypanosoma congolense (deposited in Schweizer Tropeninstitut Basel (STIB) in the form of strain No. 249) can be grown at 27°C without CO2in the medium SM/SDM 79, which contains 10% fetal calf serum and geminus. After three or four days the number of cells grows with 1×106up to 7×106/ml and the culture supernatant is separated by centrifugation, filter and concentrate the ultrafiltration. In the thus obtained culture supernatant can be installed 84% of enzyme activity, while another 16% of enzyme activity can be detected in the form associated with the cellular balance of activity. Concentrated culture supernatant used directly as a concentrate of the enzyme for reactions of TRANS-sialidase. Desired sililirovanie molecules emit after the reaction from the culture supernatant.

Example 2: Purification of the enzyme

To obtain pure enzymes concentrated culture supernatant applied to ion exchange column (Q-sepharose). Column elute after washing with salt gradients. TS2 eluted by the salt concentration to 0.2 M, TS1 gradients of salt is less than 0.2 M After elution both enzymes share using isoelectric focusing, gel filtration (Sephadex G150 SF), affinity chromatography or precipitation of the protein to apparent homogeneity.

Example 3: Determination of enzyme activity

To determine the activity of transferring the TRANS-sialidase 25 μl of an enzyme solution in 50 mm Bis-Tris buffer, pH 7.0, incubated with 1 mm Neu5Ac-α(2-3)lactose as a donor and 0.5 mm 4-methylumbelliferyl as acceptor in a final volume of 50 µl at 37°C for 2 hours. The incubation is stopped by adding 1 ml ice in the s. After the reaction mixture is applied on the pre-filled with 0.3 ml of Q-sepharose FF (acetate form) and pre-equilibrated water column. After washout of the acceptor water and throwing free volume (200 μl 1N HCl) to elute the product 1N HCl (700 ml). After acid hydrolysis of the product at 95°C for 45 minutes and cooling on ice, the sample 250-290 μl neutralized 2n NaOH and 300 μl of 1 M glycine/NaOH buffer pH of 10.0. Measure the fluorescence of free methylumbelliferone in black 96-well plates (Microfluor, Dynex, U.S.A) at a wavelength of excitation 365 nm and the wavelength of emission of 450 nm. The enzyme activity corresponds to the intensity of the measured fluorescence and can be read by pre-built calibration curve (Method Engstler et al. 1992).

Example 4: obtaining the production of transgenic organisms (bacteria, yeast, fungi, plants) in respect of this enzyme

Described here for the first time partial DNA sequence TS1 and TS2 allow using routinely used standard methods to install full-length DNA sequences of these enzymes (in particular, because here presented DNA sequences do not contain the non-coding introns). Relevant standard methods are, for example, the way "polymerase chain reaction" (PCR method), "BL is tting the Southern" includes methods for genomic DNA or cDNA and mRNA, which can be carried out using commercially available kits, for example, of firms Invitrogen or Clontech. These methods are known to the person skilled in the art and described, inter alia, in: Ausubel et al.: Current Protocols in Molecular Biology, Edition 1989 and 2001. Full-length DNA of the respective enzymes or functional partial sequence from it enter in the desired organisms-producers using standard transformation methods (Ausubel et al.). These transgenic organisms produce TS1 and TS2, and they can be isolated from transgenic organisms and/or their cultural supernatants. As organisms recipient DNA that encode TS1 and TS2, using prokaryotic bacteria, eukaryotic microorganisms, yeast and other fungi, culture of eukaryotic cells, algae, plants, seeds, animals, animal parts, tissues, hybridoma, transgenic organisms and geobiological, gene therapy and transgenic recombinants, as well as derived from these organisms, organs, tissues and cells. Stated here, the enzymes can be isolated from the corresponding whole transgenic organisms or parts thereof, of their cultural supernatant of organs, tissues, cells, biological fluids, exudates, eggs, blood, lymph, milk, plants, algae and seeds, as well as of their parts.

Example 5: the Example of the reaction of enzymes

kg glycomacropeptide (GMP) is dissolved together with 1 kg of galactooligosaccharide with a chain length of 6-10 sugar residues in commercially available 50 mm Bistro-buffer pH 7.0 (for example, Merck, Darmstadt). The solution is mixed with 1 liter containing TRANS-sialidase culture supernatant Trypanosoma congolense and cultured at 37°C for 3 hours. After this time period the TRANS-sialidase transfers sialic acid from GMP to galactooligosaccharide. Sililirovanie products can be using conventional chromatographic methods (Ausubel et al.) or technology filtration separated and purified and made clean for the preparation of compositions of this product.

Glycomacropeptide (GMP) is a byproduct of making cheese from cow's milk. After precipitation of casein for making cheese, it can be allocated from the remaining whey using filter technologies.

Galactooligosaccharide get the transformation of lactose obtained from firms of the enzyme beta-galactosidase. When this transformation of beta-galactosidase cleaves, on the one hand, the lactose to its Monomeric sugar. On the other hand, when the splitting occur in side reactions are also more dlinnostebelchatye galactooligosaccharide, which can be separated and then provided as acceptors for TRANS-sialidase reaction.

Example 6: Using enzymes

Both isolated enzyme can be used, for example, to sililirovanie containing beta is lactose polymers (such as Arabian gum, etc. and, in particular, for polylactones and galactanes, and galactooligosaccharides (GOS), in particular, for beta-galactooligosaccharides, such as, for example, Vivinal GOS company Borculo Domo Ingredients (BDI) and Oligomate 55 company Yakult.

These polymer galactose-sugar and newly formed galactooligosaccharide (receive, as described in example 5) can cialiserectile using claimed in this patent the TRANS-sialidase. As a donor of sialic acids can be used all of the above donors and, in particular, glycomacropeptide of Caseins (humans, cows, goats, sheep, horses, camels and other animals). Sililirovanie sugar patterns reveal increased similarity with acidic sugars, which can also be found in the human body and have diverse functions.

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1. Translease, which can be obtained from Trypanosoma congolense, characterized by one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, or a sequence having at least 75%identity with one of the above sequences.

2. Translease 1 (TS1) according to claim 1, characterized by at least one of the following characteristics:

The nucleotide sequence SEQ ID NO: 1
Amino acid sequenceSEQ ID NO: 2
Temperature optimum °C30-40
The optimum pH6,5-8,5
Isoelectric point, pH4-5
The molecular mass of the native electrophoresis kDa400-600
Molecular weight in pampering electrophoresis LTO-SDS page, kDa90

3. Translease 2 (TS2) according to claim 1, characterized by at least one of the following characteristics:

The nucleotide sequenceSEQ ID NO: 3
Amino acid sequenceSEQ ID NO: 4
Temperature optimum °C30-40
The optimum pH6,5-8,5
Isoelectric point, pH5-6
The molecular mass of the native electrophoresis kDa120-180
Molecular weight in pampering
electrophoresis LTO-SDS page, kDa90

4. Translease one of claims 1 to 3, originating from the organism Trypanosoma congolense.

5. Translease one of claims 1 to 3, obtained using synthetic, in particular chemical, biochemical, enzymatic, geotechnologies and transgenic methods.

6. Translease according to claim 2 or 3, amino acid sequence or amino acid partial sequence which detects relative to the corresponding amino acid sequence or partial amino acid sequence according to SEQ ID NO: 2 or 4, the sequence identity is at least 75%, calculated in accordance with the algorithm of Pearson und Lipraan, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448; and contains one or more deletions, additions, substitutions or inversions of one or more amino acid residues; and still retained the ability to catalyze the transfer of sialic acid from the donor to the acceptor.



 

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FIELD: agriculture.

SUBSTANCE: genetic modification of wheat in the form of mutation in gene SBEIIa with reduction of level of its activity.

EFFECT: grain with high content of amylose in its starch; wheat with reduced level of SBEIIb-activity with grain with non-wrinkled phenotype, with relatively high content of amylose.

62 cl, 28 dwg, 12 tbl, 16 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing a version of glycolipid acyltransferase enzyme, which involves: (a) selection of an initial enzyme which is glycolipid acyltransferase, where the enzyme contains amino acid motif GDSX, where X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S; (b) modification of one or more amino acids to obtain a glycolipid acyltransferase version; (c) testing the glycolipid acyltransferase version on transferase activity, and optionally hydrolytic activity with respect to a galactolipid substrate and, optionally, a phospholipid substrate and/or, optionally, triglyceride substrate; (d) selection of the version of the enzyme with high activity with respect to galactolipids compared to the initial enzyme; and, optionally, (e) obtaining a large amount of the version of the enzyme. The invention also relates to versions of the lipidacyltransferase enzyme, where the enzyme contains amino acid motif GDSX, where X is one or more of the following amino acid residues L, A, V, T, F, Y, H, Q, T, N, M or S, and where, compared to the initial sequence, the version of the enzyme contains one or more amino acid modifications.

EFFECT: obtaining versions of an enzyme with high transferase activity compared to the initial enzyme.

36 cl, 61 dwg, 14 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used for producing esters of such compounds as carbohydrates, proteins, protein subunits and hydroxyacids. The method involves mixing an acyl group donor, acyl group acceptor and water with formation of a medium with high water content, containing 5 to 98% water. The acyl group donor used is a lipid substrate, chosen from phospolipid, lysophospholipid, triacylglyceride, diglyceride, glycolipid or lysoglycolipid, and the acyl group acceptor used is carbohydrate, protein, protein subunitor hydroxyacid. The mixture is brought into contact with lipidacyltransferase, which catalyses alcoholysis and/or re-esterification and is an enzyme which has acyltransferase activity, containing a GDSX fragment of amino acid sequence, where X stands for one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S.

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18 cl, 51 dwg, 10 tbl, 13 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology and is a method of detoxicating mycotoxins. According to the invention, mycotoxin is reacted with glucosyltransferase in the presence of activated glucose.

EFFECT: reduction of toxicity of mycotoxins.

18 cl, 6 dwg

FIELD: technological processes; medicine.

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EFFECT: preparation of new sphingosinekinase with reduced catalytic activity.

24 cl, 15 dwg, 4 tbl, 5 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to a novel strain of microorganisms producing cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) with the enhanced specificity with respect to formation of beta-cyclodextrin. The strain of bacterium Paenibacillus campinasensis IB-10155 is deposited and stored in Collection of microorganisms in Biology Institute UNTS RAN. Activity of CGTase in cultural fluid is estimated to be 5.8 U/ml wherein 1 unit of activity is determined as amount of CGTase catalyzing formation of 1 mcM of ACB beta-cyclodextrin per 1 minute from 2% starch solution at pH = 6.0. Part of beta-cyclodextrin in sum of other cyclic products exceeds 70%. Invention provides high stability of CGTase catalyzing the transformation reaction of starch to beta-cyclodextrin at relative constant part of this product to sum of other cyclic dextrins in early steps of reaction.

EFFECT: valuable properties of strain.

2 tbl, 4 ex

FIELD: plant gene engineering and food processing industry.

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31 cl, 12 dwg, 3 tbl, 2 ex

FIELD: organic chemistry, chemistry of polymers.

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EFFECT: improved and valuable properties of block-copolymers.

11 cl, 78 ex

FIELD: biotechnology, peptides, genetic engineering.

SUBSTANCE: invention relates to constructing nucleic acid molecule comprising functional in starch-containing plant tissues promoter, fragment encoding transit peptide for translocation of useful peptide into amyloplast, fragment encoding useful peptide, region encapsulating into starch and terminator. In insertion into plant genome DNA molecule expresses hybrid polypeptide comprising the desirable protein encapsulated into starch matrix. Prepared vegetable material can be used, for example, in manufacturing fodders for mammals, fishes and poultries. Also, invention can be used in food industry.

EFFECT: valuable properties of nucleic acid and polypeptides.

15 cl, 19 dwg, 9 tbl, 7 ex

FIELD: biotechnology, in particular method for construction and production of mutant transglutaminases (MTG).

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6 cl, 60 dwg, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology, animal husbandry and medicine. Proposed is a transgenic animal from the family of bulls which produces in its milk a recombinant human growth hormone in amount of at least 92 ng/ml per month on average. Also disclosed is a method of creating such a transgenic animal which involves cloning the genetic make-up which codes the hGH gene and a beta-casein promoter in an expression vector. Further, there is transfection into fetal somatic cells of cattle, usually fibroblasts, and nuclear transfer into enucleated oocytes of cattle, that way forming transgenic embryos. The invention can be used in animal husbandry and medicine.

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43 cl, 5 dwg, 3 tbl, 9 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to biochemistry and specifically to a modified polypeptide of a HIV-1 gp41 envelope glycoprotein, a polynucleotide which codes the modified polypeptide and an expression vector which contains a coding modified polypeptide of the HIV-1 gp41 envelope glycoprotein. The modified polypeptide of the HIV-1 gp41 envelope glycoprotein contains an amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 14, where the sequence between positions 603 and 615 or 598 and 622 SEQ ID NO: 1 or the sequence between positions 530 and 542 or 525 and 549 SEQ ID NO: 14 is replaced by a linker fragment which is an oligopeptide SEQ ID NO: 2.

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10 cl, 8 dwg, 1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: integrative plasmid vector contains selective marker for selection of transformants in cells E.coli, site of replication start, site of integration and expression cassette, which consists of promotor, transcription terminator and selective marker, and integration site is represented by sequence of DNA that codes area 18S of messenger RNA.

EFFECT: improved productivity of strain.

3 cl, 4 dwg, 10 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents polypeptide, having α-L-arabinofuranosidase activity selected from the following polypeptides: polypeptide with SEQ ID No. 2, polypeptide, amino-acid sequence of which is located between positions 28 and 400 SEQ ID No. 2, fragment of polypeptide with SEQ ID No. 2, having activity of α-L-arabinofuranosidase, polypeptide having activity of α-L-arabinofuranosidase B and expressing 80% identity with polypeptide SEQ ID No. 2. Invention also relates to polynucleotide, which codes this polypeptide, expression cassette and vector, containing polynucleotide, and master organism that contains this polypeptide.

EFFECT: expanded arsenal of mediums for hydrolysis of α-L-arabinofuranosyl links in arabinofuranosyl-oligosaccharide compounds.

9 cl, 6 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: genetic makers of siSTRIKE-neo vector producing interfering RNA (siRNA), are inhibitors of reproduction of human immunodeficiency virus type 1. Invention allows producing effective anti-HIV preparations of siRNA produced in cells by the administered genetic makers containing palindrome intended for formation of siRNA production and selected with using non-virus and virus models.

EFFECT: invention can be used in medicine and researches.

12 cl, 3 dwg, 3 tbl, 3 ex

Product and method // 2385931

FIELD: medicine.

SUBSTANCE: invention concerns biotechnology and represents a new alpha galactosidase, a DNA molecule coding it. Besides the invention concerns an expression vector containing such DNA molecule, and also a cell transformed by the vector. The invention also concerns the method for making alpha-galaktobiose disaccharides with using new alpha galactosidase.

EFFECT: invention allows for high-efficient alpha-galaktobiose disaccharides.

15 cl, 5 dwg, 1 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is a method of producing staphylokinase using an OXY-1 cassette with sequence SEQ ID No. 1. The cassette is part of two plasmids with international inventory numbers BPL-0020 and BPL-0021, which are transformed strains of bacteria E. coli for producing staphylokinase.

EFFECT: possibility of obtaining highly effective staphylokinase.

16 cl, 11 dwg, 4 ex

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