The selected nucleic acid, plasmid, a replicable expression vector, transformed cell line mouse 127, polypeptide, the method of obtaining a precursor polypeptide having scce activity, a pharmaceutical composition for treating disorders associated with keratinization

 

(57) Abstract:

The invention relates to biotechnology and medicine and can be used to produce recombinant chymotrypsinlike enzyme corneal layer (SCCE). Recombinant polypeptide having SCCE activity, obtained by culturing murine cells Is transformed by the expression vector containing the nucleotide sequence (SEQ IDN1), coding for the specified polypeptide. In the pharmaceutical composition for treating disorders associated with keratinization include 0,00 l - 25% native or recombinant S. The invention allows to obtain the enzyme active against the corneal layer of the skin, and its use in pharmaceutical and cosmetic compositions. 7 S. p. f-crystals, 23 ill., 5 table.

The invention relates to the recombinant polypeptide to a nucleotide sequence that encodes a polypeptide, the expression system that can Express the polypeptide, as well as to pharmaceutical and cosmetic compositions containing the polypeptide, and the use of the polypeptide for various cosmetic or therapeutic purposes.

Brief description of the invention

The skin as an organ, predstavili, which are either organ-specific, such as psoriasis and eczema, or manifestations of such common diseases as common allergic reactions. The fact that there are cospecification disease, can be considered as a proof of the existence of molecular mechanisms that are unique to the skin. Similarly, research cospecification molecular processes are important for the understanding and treatment of skin diseases. Apparently, it is reasonable to assume that some of these processes, one way or another, associated with the specialized functions of the skin, that is, with the creation of a physical and chemical barrier between the organism and the external environment. Physico-chemical skin barrier localized in the upper layer of the skin, corneal layer (Stratum corneum).

The corneal layer is the most specialized structure of the skin. It is the final product of the processes of differentiation of the epidermis, i.e., stratified squamous epithelium, which is the upper part of the skin. Most cells of the epidermis consists of keratinocytes at various stages of differentiation. The most deeply located keratinocytes, basal cells, are baseline only keratinocytes, having the ability to divide. Part of the basal cells constantly leaves basal membrane and undergoes a process of differentiation, in which cells become the building blocks of the corneal layer. In this process, keratinocytes undergo a series of adaptive changes. There is an increase in the content of the cytoskeleton, consisting of the epidermis-specific cytokeratins. Intermediate filaments adjoining cells are connected with functional fragments due to the increasing number of desmosomes. The most dramatic changes occur during the transition from the uppermost layer of living cells, stratum granulosum, the dead corneal layer, stratum corneum, in the process, which usually is called keratinization. Covalently associated proteins are located close to the inner part of the plasma membrane, forming a very stable cellular membranes. In addition, rich lipid substance, formed in the organelles keratinocyte-specific cells, is secreted in the extracellular space, and forming lipid lamellae that surround the cells of the corneal layer is a barrier permeable to hydrophilic substances. And finally, all of the intercellular structure, except for densely packing what time cells. This means that the regulation of various processes in the corneal layer must be the result of "programming" on the stage when the keratinocytes are still living cells. Transformation of the epidermis, which usually occur within about four weeks, and the cells are part of the corneal layer for approximately two weeks, will result in discontinuation of cells from the surface of the skin during exfoliation /desquamation/. This process is an example of "programming" the corneal layer. A necessary condition for the functioning of the corneal layer as a physical and chemical barrier is the requirement that the individual cells are held together due to the mechanically stable structures, that is, of desmosomes. Degradation of desmosomes, which is necessary for desquamation, should be regulated in such a way as to ensure that the peeling from the surface of the skin, which would be balanced by the formation of a new corneal layer, without disrupting the barrier function of the tissue.

Violation of keratinization

In the basis of a large number of pathological conditions of the skin of varying severity is a violation of the process of keratinization. When psoriasis occurs in addition to the typical chrons this disease peeling. There is a group of congenital skin diseases characterized by thickening of the corneal layer, which leads to the formation of "fish scales" so-called ichthyosis. Some individuals suffering from ichthyosis, observed reduced rate of desquamation. Though less serious than iktinos, disease "dry skin" /pigmentosum/ also characterized by corneal layer, which sloughed off the corneocytes, not, as in normal conditions, in the form of individual cells or small aggregates of cells, but in the form of large, macroscopic scales; this disease is quite common for the elderly, but also among atopic individuals with lowered resistance to skin lesions and susceptibility to the development of distinctive forms of endogenous eczema. In the case of acne disease is observed disturbed keratinization in the ducts of the sebaceous glands, which leads to the formation of comedones in the blockage of the glands. The formation of comedones progresses and, it is believed, leads to the formation of inflammatory acne sores. Proteolytic enzymes are involved in keratinization.

There are several stages in the process of keratinization and during transitions corneal layer, where proteolytic enzymes, in hatinovich threads, observed in the transition from live to dead epidermal layers should include proteolysis. Turning profilaggrin in filaggrin, protein, which is thought to function in a specific type of aggregate filaments of cytokeratin in the process of keratinization may kataliziruetsa specific proteinase. In the corneal layer of filaggrin further decomposed to low molecular weight components, which are probably important as natural moisturizers". In addition, there are proteolytic modification cytokeratin polypeptides in the process of keratinization. Finally, proteolytic acts, apparently, play a crucial role in the degradation of intercellular cohesive structures in the corneal layer in the processes that normally lead to desquamation.

Cohesion and desquamation of cells of the corneal layer

/Stratum corneum/

The role of desmosomes

Intercellular cohesion in the corneal layer, as well as in living parts of the epidermis, largely at the expense of desmosomes. Desmosome consists of two symmetrical halves, each of which is formed by two contiguous cells. Each half of desmosome has one intracellular part associated with filaments of cytokeratin and admi. The extracellular portion of these proteins, desmoglein represent adhesion molecules, and due to their interaction with each other in the extracellular space is formed cohesive structure. Degradation of desmosomes, apparently, runs several other ways in the corneal layer of the palms and soles /feet/ compared with the corneal layer, not related to the palms soles. In the latter tissues around 85% of desmosomes disappears shortly after the cells are completely dead. The remaining desmosome, which are, preferably, villous on the edges of extremely flattened cells, appears to remain intact up to the level where desquamation. In the corneal layer, which does not apply to palms and soles, the corneocytes are much less flattened, and there is no degradation of desmosomes in the deeper layers of tissue. In both types of tissues desquamation associated with the degradation of desmosomes. If ational skin, as in the case of "dry skin", the number of desmosomes in the superficial layers of the corneal layer, as shown, increases.

The intercellular lipids of the corneal layer

Differences in the degradation of desmosomes relating to the palms and soles of the corneal layers and not adaderana lipids significantly higher in the corneal layers, not related to the palms soles. As a result, the efficiency of this fabric as a barrier permeable to water and other hydrophilic substances is much greater than the permeability of the corneal layer of the palms and soles. As desmosome occupy a significant amount and as intact desmosome prevent widening of the intercellular space, namely the degradation of desmosomes may be the mechanism by which larger extracellular space becomes available for lipids. Extracellular lipid corneal layer associated with desquamation and also some other ways. Because they form the bulk of the extracellular space, it can be expected that they have a significant impact on the activity of enzymes that act in this place, for example, enzymes responsible for the degradation of desmosomes. Indeed, various disorders of lipid metabolism, as has been shown, apparently, cause some types of ichthyosis. Apparently, also, the lipids in some degree contribute to the cellular cohesion corneal layer. Moreover, the secretion of lipids in the intercellular space of the corneal layer, appears to be associated with the secretion of several enzymes. Predecessors lipids are stored as shown, contain a number of hydrolytic enzymes. Thus it is assumed that the enzyme responsible for the degradation of desmosomes in the corneal layer, is synthesized, it is possible in an inactive Pro-form, keratinocytes, is stored in lamellar bodies and secreted into the intercellular space of the corneal layer in the process of keratinization, where it can be activated, and its activity is further regulated by, among other factors, extracellular lipids.

Disorders of cellular cohesion in the viable epidermis

There are a number of skin diseases that violate the cohesion between keratinocytes in neurogenetic viable epidermal layers. These diseases are characterized by a phenomenon called acantholysis, i.e. destruction desmosome contact between keratinocytes, which in other forms seem to be normal. This process, apparently, at the expense of proteases, which are still not been identified. Quantales, in its acute forms, leads to the formation of blisters and is a characteristic of the autoimmune disease pemphigus vulgaris and pemphigus foliaceus hereditary benign familiar pemphigus /disease Hayley-Hayley's/ and hereditary dyskeratosis follicularis /¾

In addition to his functions as creating a physical and chemical barrier between the internal body and the external environment, the epidermis also functions as an active immunological barrier. Keratinocytes have the ability to produce and regulate a large number of cytokines and other mediators of inflammation, due to which they commute and interact with cells of the immune and inflammatory systems. It is of great importance in protecting the host from microbial infection and wound healing. Modern research has also shown that keratinocytes, apparently, actively involved in many inflammatory skin diseases such as psoriasis and eczema.

Epidermal proteases may play an important role in inflammatory processes

One of the cytokines produced by keratinocytes, is interleukin 1 /Il-1/. Il-1 exists in two forms, Il-1 and Il-1 , and both are present in the epidermis. If Il-1 after synthesis is fully active, Il-1 is synthesized as an inactive 31 KD Pro-forms. Pro - Il-1 becomes active 17 KD form due to specific proteases present, for example, in monocytes, but still not on the waste /fecal pancreatic and cathepsin G neutrophilic granulocytes/ maybe however, to serve as activators of Pro - Il-1 .

As suggested by Norris /Norris 1990/, proteolytic enzymes present in the intercellular space of the corneal layer, can in certain circumstances be able to convert the inactive form of cytokines in active form. Of particular interest in this context is biologically inactive Pro-interleukin-1 , which, as shown, is produced by keratinocytes /Migutani et at., 1991/. Still not been discovered enzymes with the ability to convert a Pro-interleukin-1 in the active interleukin-1 . However, as chymotrypsin and cathepsin G /chymotrypsinogen enzyme/ have the ability to catalyze the conversion of inactive 31 KD recombinant Pro-interleukin 1 in a fully active 17 KD form, it is possible that epidermal chymotrypsin-like enzymes can catalyze this transformation.

Detailed description of the invention

The present invention relates to enzyme, which was named chymotrypsinogen enzyme strarum corneum /corneal layer// SCCE/, which is regarded as the enzyme responsible for the degradation of desmosomes in the corneal layer. The evidence presented in example 1, demonstrates that exfoliation kletenik for this enzyme, apparently, is chymotrypsin-like serine proteinase, which can inhibit zinc ions.

Example 2 describes the opening chymotrypsinlike enzyme Stratum corneum /SCCE/, proteases that satisfies the criteria responsible for the degradation of intercellular cohesive structures in the corneal layer in vitro and possibly also in vivo. Example 3 describes a partial characterization of the activity chymotrypsinlike enzyme stratum corneum /SCCE/ using chromogenic substrates.

The results obtained demonstrate that SCCE different Entomologicheskoe from the rest chymotrypsinogen proteases. The nature of inhibition and the ability to decompose two different substrate for trypsin-like enzymes is significantly different for SCCE compared with bovine chymotrypsin and cathepsin G of human rights. Apparently, SCCE also differs from chymase mast cells. The latter enzyme catalyzes the degradation of Suc-Ala-Ala-Pro-Phe-pNa now /see Schwartg et al., 1987, and Schelter et al., 1989/ - what does SCCE - and not inhibited by Aprotinin SBTJ /Shelter et al., 1983 and Wintroub et al. 1986/, whereas SCCE inhibited.

Example 4 describes the partial purification of SCCE KCl extracts of corneocytes using affinity chromatography on resolubilize unrestored samples were obtained good outputs in stages 1-6, but decreased to 0 at the stages 7 and 9, and outputs to the subsequent stages were significantly reduced. For the reduced samples at stages 7 and 9 are not detected derivatives of amino acids, but at the subsequent stages, which detected derivatives, the sharp fall in output was observed. These results suggest that in positions 7 and 9 are Sistani. However, it was not possible to determine karboksimetilirovaniya cysteine at stages 7 and 9 after the recovery processing Vodokanal acid /100 mm/. The obtained sequence /Fig. 13, the placenta. ID N 3/ was identical for both the restored and unrestored samples.

Example 5 describes obtaining SCCE-specific monoclonal antibodies and polyclonal SCCE-specific antibodies chicken and rabbit, as well as immunohistochemical study with monoclonal antibodies.

Example 6 describes the cloning and sequencing of cDNA encoding human SCCE. First, cDNA library, obtained on mRNA obtained from epidermally keratinocytes adult, sceneroot anti-SCCE rabbit polyclonal antibody. One of these antibodies gives a high background signal, and the second antibody /D-5/, the number of immunoreactive plaques enrich as privacidade true-positive plaques. Was not observed reactivity with monoclonal antibodies oAb 4 and oAb 9 for none of the plaques. Intensive characterization of restriction enzymes and characterization of PCR eleven selected plaques did not identify any similarities between the different plaques. On the basis it is impossible to obtain a "fingerprint" probable cDNA sequence SCCE, the strategy had to be modified.

Despite the preferred detection SCCE immunoreactive material in suprabasally the keratinocytes, for screening cDNA SCCE used a cDNA library derived from cultured human keratinocytes. It can be expected that this library should contain cDNA only from basal keratinocytes. This attempt was based on the observation of weak, but probably significant immunostaining using SCCE monoclonal antibodies from basal keratinocytes.

Plaques were skanirovaniya using synthetic 17-dimensional oligonucleotide probe designed on the basis of the most reliable part of the amino acid sequence Ile-Ile-Asp-Gly-Ala-Pro /polenske, as described in example 4. Because of the uncertainty within the experimentally defined amino acid sequence, this Hexapeptide, as it represents one of the most reliable parts. In addition, the possible codons encoding the Hexapeptide, lead to the smallest possible degeneration of the DNA probe. Longer experimentally determined sequence Gln-Val-Ala-Leu-Leu-Ser-Gly-Asn-Gln-Leu /sequence ID No. 3, aa 15 aa 24/ was excluded because of the high degree of degeneration of the sequence encoding this peptide sequence. Fourteen positive plaques were identified during the initial skanirovaniya. These positive plaques were again skanirovaniya using the same probe and method that have been described previously. After the procedure, re-screening identified two positive plaques. These two selected plaques were purified, and the size of the inserts was determined by PCR using SY 1600 and SYM 1601, which was complementary to the two arms of the phage, as primers, and the selected phages as matrices. This cloned fragment was subjected to partial sequence analysis.

Broadcast received DNA poseidonos protein sequence. However, this sequence was not translational start codon. For isolation of full length cDNA, the DNA fragment was isolated on an agarose gel and used as a probe, providing hybridization in tough conditions. To obtain full length cDNA, a cDNA library was rasklinivanie twice with the probe, using the same methods that were previously described, except that the hybridization was led to stringent conditions at 65oC. These experiments led to the identification and allocation of 45 individual positive plaques that were initially skanirovaniya in PCR analysis using SYM 1600 or SYM 1601 in combination with SYM 3208 as PCR primers for the identification of plaques containing the complete 5' open reading frame.

After further screening and sequencing of the obtained PCR amplificatoare fragments obtained from these phages were cloned, as was described in example 6, and the obtained results indicate that one phage 205.2.1 contains full length insert. Was determined the complete nucleotide sequence of the cDNA fragment. The nucleotide sequence /sequence ID No. 1/ contains an open reading frame sufficient to cod, including the signal peptide and propolypeptide /sequence ID No. 2/.

Example 7 describes the definition in the human epidermis two SCCE mRNA species that are able to gibridizatsiya with cDNA probes derived from the SCCE cDNA sequence.

Example 8 describes the expression of recombinant SCCE in E. coli. The result shows that it is possible to produce recombinant SCCE in bacteria.

Example 9 describes the expression of recombinant SCCE person in mammalian cells. Get three protein that demonstrate the reaction with all viable polyclonal antibody SCCE rabbit and chicken, as well as the deposited monoclonal antibody. Recombinant proteins that are reactive with respect to antibodies generated against native SCCE demonstrate the apparent molecular weight, which is about 1 KD greater than that of native purified human SCCE. Recombinant protein does not show any proteolytic activity.

The selection, activation and further characterization of recombinant SCCE described in example 10. The inactive Pro-form of recombinant SCCE can be activated proteolytic pepcid after basic amino acids, such as endoproteinase LYS-C, papain and plasmin may be able to activate Pro - SCCE in active SCCE. It was found that the signal peptide consists of 22 amino acids and is based on the N-terminal amino acid sequence of the native active SCCE, propeptide, consisting of seven amino acids. Moreover, it was shown that the obtained recombinant SCCE exists in two N-glycosylated forms and one deglycosylation form, which is similar to results obtained for active native SCCE.

The term "stratum corneum" chymotrypsinlike enzyme /SCCE/" /chymotrypsinlike enzyme corneal layer or polypeptide having SCCE activity" in the broad sense of the mean serine prosthesis or its Pro forma, which can be activated through proteolytic cleavage, and the specified enzyme in its active form is inhibited by the same inhibitors and in exactly the same way as spontaneous cell dissociation that can be called in the model system with samples of the Horny layer of the skin, encourageme at neutral or close to neutral pH at physiological temperatures.

More specifically, the term "polypeptide having SCCE activity" defines Tobin decomposing MeO-Suc-Arg-Pro-Tyr-pNA /S= 2586/, moreover, this decomposition of the polypeptide is inhibited by Aprotinin, hemostasia and zinc sulfate practically by way of examples 3 and 13, as illustrated in Fig. 9 and in table 5.

More specifically, the term "polypeptide having SCCE activity" includes a polypeptide that is able to induce proteolytic cleavage of proteins of desmosomes - desmoglein 1 during in vitro incubation of the corneal layer of the heel part.

Such a polypeptide is typically also reacts with antibodies generated against native SCCE, which was isolated from an extract of dissociated cells of the corneal layer of the heel surface. Examples of such antibodies are polyclonal antibodies, obtained as described in 5.2, and monoclonal antibodies TE4b and TE9b, obtained as described in example 5.1. Monoclonal antibodies produced by hybridomas TE4o and TE9o deposited in the European collection of animal cell cultures, Porton Down, Salisbury, Wiltshire SR 4 OJP, United Kingdom under registration numbers ECACC 93061817 and EC ACC 93061816, respectively, in accordance with the requirements of the Budapest agreement.

Cloning and sequencing of cDNA encoding human SCCE described in example 6. Nucleotide posledovatelnosti protein precursor SCCE, consists of 253 amino acids, including the signal peptide and propolypeptide, was thus found. The nucleotide sequence presented as sequence ID No. 1, and the resulting amino acid sequence "chymotrypsinlike enzyme corneal layer /SCCE/" is presented as sequence ID No. 2.

The term "Pro - SCCE or its analogue or variant of his" imply a polypeptide containing the amino acid sequence ID No. 2 or an analogue or variant sequence when the nucleotide sequence of the invention is expressed in a suitable expression system, and which, after proteolytic activation leads to the formation of serine proteases, which can inhibit the same inhibitors that spontaneous cell dissociation, which can be called in the model systems with samples of the Horny layer of the skin, encourageme at neutral or nearly neutral pH at physiological temperatures, i.e. about 37oC. in General, the protein will react with antibodies produced against purified native or recombinant SCCE.

The term "SCCE or its analogue or variant of its" podrazumevalo, which is produced when the nucleotide sequence of the present invention is expressed in a suitable expression system, and which is a serine-protease, which can inhibit the same inhibitors that spontaneous dissociation of cells that can be induced in model systems with samples of the keratinized layers of the skin, inkubiruemykh at neutral or nearly neutral pH values at physiological temperatures, i.e., about 37oC. in General, the protein must react with antibodies generated against native or recombinant SCCE.

The term "analogue or variant" means a polypeptide, the sequence of which does not exactly match the amino acid sequence ID No. 2, but which still retains the "SCCE activity", as defined previously. Generally, such polypeptides are polypeptides that differ, for example, to some extent on the composition of amino acids or include post-translational modifications, e.g. glycosylation or phosphorylation, compared with the SCCE protein described in the examples.

The term "analog" or "variant" is used in this context for oboznachaiushchaia the amino acid sequence ID No. 2, obtained from the SCCE protein as described in example 6, allowing small variations that change the amino acid sequence, for example, deletions, site-directed mutagenesis, insertion of additional amino acids, or combinations thereof, to create SCCE protein analogues. These modifications can lead to inverse and useful new properties similar. Analog polypeptides or proteins can be obtained from animals or humans, or they may be partially or fully synthesized. Analogs can also be obtained by using techniques of recombinant DNA.

Thus, an important variant of the present invention refers to a polypeptide in which at least one amino acid residue is replaced with another amino acid residue and/or in which at least one amino acid residue was removed or added, so that the resulting polypeptide containing the amino acid sequence that differs from amino acid sequences shown in sequence ID No. 2, or suppositionally specified amino acid sequence, as defined herein, but practically having SCCE activity, as indicated previously.

Interpeptide of the present invention, containing from 50 to 250 amino acids, for example, at least 70 amino acids, at least 100 amino acids, at least 150 amino acids, or at least 200 amino acids.

The most important variant of the invention is a polypeptide containing the amino acid sequence-7 - 224 in sequence ID No. 2 /Pro-SCCE/ and the polypeptide containing the amino acid sequence 1-224 in sequence ID No. 2 /SCCE/.

The term "enzymatically active substance" denotes a polypeptide sequence that contains only part of the polypeptide sequences shown in sequence ID No. 2, and which has the enzymatic activity. This includes polypeptide suppositionally that were analgesicany due to modifications, as is indicated here. Specific polypeptide or polypeptide which has the enzymatic activity, is especially interesting.

The predicted amino acid sequence ID No. 2 was compared with amino acid sequences of the enzymes chymotrypsin person /Joulta et al., 1989/, cathepsin G man /Salvesen et al., 1987/ and chymase mast cells human /Caughey et al., 1991/. Although the predicted amino acid is very low, about 33-38%. In this respect it seems that the SCCE has only moderate similarity to previously known serine proteases. On the other hand, SCCE is a typical serine-proteinase against his-tag, aspartic and serine residues of the active sites and conservative sites located near these sites. This is true also for most of the cysteine residues and other highly conservative areas of serine proteases. In the depth of the pocket /pouch/ primary specificity /residue 189 chymotrypsin/ is serine residue fecal human chymase mast cells and prostate-specific antigen and alanine residue cathepsin G of human rights. In SCCE, on the other hand, this position is occupied aspartic residue. This may explain the discovered fact that although SCCE undoubtedly has chymotrypsinogen activity, its relative activity towards various chromogenic peptide substrates differs from that of chymotrypsin and cathepsin G, as well as inhibiting the effectiveness of hemostatic, low molecular weight inhibitor of the chymotrypsin-like enzymes.

Comparison of sequences of fecal human cathepsin G of human rights and chymase tochnit these sequences, at the end of this description.

A comparative analysis of primary structures is carried out manually.

Legend:

HUMCTRP = chymotrypsin person /Jouita et al., BBRC 158:569-575, 1989/

HUMCHTG = cathepsin G man /Salvesen et al., Biochemistry, 26:2289-2293, 1987/

HUMCHYM = chymase mast cells human /Caughey et al., J. Biol. Chem., 226:12956-12963, 1991/

Gomologichnosti:

HUMCTRP/SCCE: 85/224 = 38%

HUMCHTG/SCCE: 74/224 = 33%

HUMCHYM/SCCE: 74/224 = 33%

HUMCHTG/HUMCHYM: 109/226 = 48%

The numbering refers to the chymotrypsinogen

Underlined:

Conservative in the vicinity of the active site /Ile 15, His 57, ASP 102 and Ser 195 chymotrypsin/ pockets and primary specificity of chymotrypsin /Ser 189, Ser 213 - Trp 215, Gly 226 chymotrypsin/.

Stars: possible sites of N-glycosylation in SCCE.

Thus, an important variant of the present invention refers to a polypeptide containing the amino acid sequence in which consecutive string of 20 amino acids homologous, at least 80% of the threads of the amino acids of the same length, selected from the amino acid sequence ID No. 2.

Polypeptide sequence of the present invention, homologous, at least 80%, such as 85% or at least 90%, polypeptide, presented as sequence ID No. 2, apparently, completely unique in the scope of the present invention includes polypeptides for which the degree of gomologichnosti with similar sequential thread of the 20 amino acids selected from the amino acid sequence ID No. 2, may not be more than 55%, but preferably not more than 70%. Such sequences can be obtained from similar proteins from other species, such as other mammals, like mice, rats, rabbits, Guinea pigs, pigs or cows. As a small part of the sequence may exhibit significant similarity with other serine-proteases, the scope of the present invention also includes peptides with the degree of gomologichnosti at least 95%, and most preferably at least 99% of gomologichnosti with similar sequential thread of the 20 amino acids selected from the amino acid sequences shown in sequence ID No. 2.

The term "gomologichnosti sequence" mean identity in amino acid sequence in segments of two or more amino acids in a pair with respect to the identity and position of the amino acid polypeptides.

Coleoptiles this polypeptide and the amino acid sequence, presented in sequence ID No. 2. Amino acid sequence to be compared with the amino acid sequence represented by sequence ID No. 2 can be deduced from the nucleotide sequence, such as DNA or RNA sequence, for example, obtained by hybridization, as will be described later, or can be obtained by conventional methods amino acid sequencing. The degree of gomologichnosti preferably determined on the amino acid sequence of the Mature polypeptide, i.e., without accounting for any leader sequence. Typically use only the coding region when compared nucleotide sequences to determine their internal gomologichnosti.

In this context, the term "polypeptide that is recognized by at least one of the deposited antibodies" should include the amino acid sequence that includes amino acids that make up almost consistent thread in linear or spatial conformation of any sequence of the polypeptide presented in sequence ID No. 2, which is detected by at least one of the deposited antibodies. As is well known the EIT properties and can be epitopes. The deposited antibodies TE4b and TE9b, apparently, recognize conformational epitopes SCCE.

It was shown that polyclonal rabbit antibodies obtained by the method of example 5.2.2, provide granular staining of the stratum granulosum and rather diffuse staining of the lower stratum corneum /corneal layer/ observed by immunofluorescence microscopy.

Antibodies generated against purified native or recombinant SCCE, usually associated with the upper basal cells keratinized /Horny/ squamous epithelium person, but not epithelial cells neurogenesis squamous epithelium. These antibodies also bind to the intercellular space of the Horny layer of human skin.

Antibodies reactive with the polypeptides of the present invention, suitable for use for various purposes, as are listed below:

For separation of proteins: Antibodies can be used to highlight polypeptide /polypeptide/ or its equivalent from biological samples using affinity chromatography or immunoassay.

For diagnosis and therapy: Monoclonal antibodies against the polypeptide /polypeptide/ or practical agent can be an antibody with specificity for the polypeptide of the present invention. This antibody can be linked to another protein or solid carrier and/or can be used in texts agglutination or tests manifestations color. Such antibodies can also be used for the quantitative determination of SCCE peptides or their analogs in biological samples using standard techniques of histochemistry and immunochemistry.

In one aspect the present invention relates to a nucleotide sequence that encodes a polypeptide of the invention, as defined previously. In particular, the invention relates to a nucleotide sequence containing virtually the sequence presented in the sequence ID No. 1. Other important variants refer to a nucleotide sequence that encodes a polypeptide containing suppositionally amino acid sequence ID No. 2, such as a nucleotide sequence that encodes a polypeptide containing the amino acid sequence-7-224 sequence N 2, or a polypeptide that contains the amino acid sequence 1-224 sequence ID No. 2.

The present invention relates also to a nucleotide sequence that hybridizes with nucleoli is 7 and in example 9.

In another aspect, the present invention relates to a nucleotide sequence containing the nucleotide sequence represented by sequence ID No. 1, or its analogue or her suppositionally, which

1/ homologous sequences presented in the sequence N 1, at least 90% and/or

2/ encodes a polypeptide, amino acid sequence which is at least 80% homologous to the amino acid sequences shown in sequence ID No. 2 and/or

3/ encodes a polypeptide that is bound monoclonal antibody produced hybridoma cell line TE4b that is deposited on June 18, 1993, at the ECACC under registration number ECACC 93061817 or a monoclonal antibody produced hybridoma cell line TE9b that is deposited on June 18, 1993, at the ECACC under registration number 93061816, and/or

4/ encodes a polypeptide that is bound polyclonal anticorodal produced against native SCCE, which was isolated from an extract of dissociated cells of the corneal layer of the heel surface.

In the scope of the present invention also includes a modified nucleotide posledovatelnosti excluded, substituted or modified, or at least one additional nucleotide is built so that the resulting nucleotide sequence that encodes a polypeptide having SCCE activity.

In the present description and the claims the term "suppositionally" refers to a sequence, the preferred size which is at least 15 nucleotides, more preferably at least 18 nucleotides, and most preferably at least 21 nucleotides. In some variants of the present invention suppositionally or similar nucleotide sequence of the present invention contains at least 48 nucleotides, for example at least 75 nucleotides, or at least 99 nucleotides. "Suppositionally" must meet at least one criterion 1/-4/ above or should gibridizatsiya with the nucleotide sequence represented by sequence ID No. 1.

It is well known that small fragments suitable for PCR methods as described herein. Also fragments and sequences can, among other applications, to be used as probes in the identification of>The term "analog" in relation to DNA fragments of the invention is to indicate a nucleotide sequence that encodes a polypeptide that is identical or almost identical to the polypeptide encoded by the DNA fragment of the invention. It is well known that the same amino acid can be encoded by different codons, and the use of the codon is associated, among others, with the preference of the considered organism expressing the nucleotide sequence. For example, one or more of the nucleotides or codons of the DNA fragment of the present invention may be replaced by others that are being expressed, result in a polypeptide that is identical or almost identical to the polypeptide, codereuse consider DNA fragment.

Also, the term "analog" is used in the context of the present invention to denote a DNA fragment or DNA sequences similar nucleotide composition or sequence as the characteristics of the DNA sequence ID No. 1, amino acid coding sequence, SCCE component polypeptides, providing a small

variations that do not have a significant adverse impact on the enzymatic AK is sustained fashion harmful effects" mean that the enzymatic activity equivalent should be at least 50%, more preferably at least 60%, even more preferably at least 70%, e.g. at least 75% of the enzymatic activity of native SCCE, if it is determined, for example, by the method of example 3. Similar to DNA sequence or DNA fragment can be obtained from such a body, as for example, the animal or human body, or to obtain a partially or completely synthetic way. Similar can also be obtained by using techniques of recombinant DNA.

Moreover, the terms "analog" and "sequence" should include such variations in sequences as substitution, insertion /including introns/, additions, and reconstruction of one or more of the nucleotides, and these variations should not exert any harmful influence on the polypeptide encoded by the DNA fragment or suppositionally.

The term "substitution" means the replacement of one or more of the nucleotides in the complete nucleotide sequence of one or more of the excellent nucleotides, "adding" means adding one or more nucleotides at either end of the full nucleotide placenta is of the sequences; "deletion" means that one or more of the nucleotides deleted from the complete nucleotide sequence or at either end of a complete sequence, or from any suitable point, and "rearrangement" means that two or more nucleotide residue reversed within DNA or polypeptide sequence, respectively. The DNA fragment may, however, also be modified as a result of mutagenesis, either before or after its introduction into the body.

The terms "fragment", "sequence", "suppositionally" and "analog" in the sense used in this description and the claims in respect of fragments, sequences, suppositionally and analogues in accordance with the present invention should, of course, be construed as not containing these things in their natural setting, but existing rather, for example, is selected, purified, in vitro or recombinant form.

In one embodiment of the present invention, detection and/or quantification SCCE polypeptide mRNA can be obtained by extracting RNA from cells or tissues and turning it into cDNA or subsequent use in polymerase Cenacolo, as the DNA fragment is presented in sequence ID No. 1. This method for detection and/or quantification can be used as a diagnostic method for diagnosis of pathological States in which SCCE mRNA is expressed in larger or smaller amounts than usual.

In the scope of the present invention also includes a diagnostic agent containing a nucleotide probe that can detect the nucleotide sequence of the invention and the method for the diagnosis of diseases in which CCE expression has been violated, and/or diseases in which SCCE gene mutated; and the method includes carrying out PCR analysis of a sample obtained from a patient suspected of having a disease in which there are a higher number of SCCE than usual, or are mutated forms of SCCE in which the sample is subjected to contact with the diagnostic agent as described previously, providing a nucleotide sequence able to be amplified and determining the existence of any identical or homologous nucleotide sequences in the sample.

The polypeptides of the present invention can be obtained by using recombinational sequence of the present invention.

The body, which is used to produce the polypeptide of the present invention, may be higher organism, e.g. an animal, or lower body, for example, a microorganism such as E. coli. Regardless of the type of organism, the DNA fragment of the present invention is injected into the body, either directly or using a suitable vector. In another embodiment, the polypeptides can be obtained in cell lines mammals, introducing a DNA fragment or analogue, or suppositionally of the present invention either directly or by using an expression vector.

DNA fragment or analog, or suppositionally can also be cloned into a suitable stable expression vector, and then enter into an appropriate cell line. The cells producing the desired polypeptide, then selectrow levels of productivity under conditions suitable for the vector or cell lines. Selected cells are then align forth, and they form a very important and continuous source of desired polypeptides.

A specific example of similar DNA sequences of the present invention is a DNA sequence that includes DNA serial is Essie in E coli. This DNA sequence is a sequence which, when embedded in E. coli together with a suitable regular sequence, leads to expression of the polypeptide containing virtually amino acid sequence represented by sequence ID No. 2, or a part of it. Thus, the DNA sequence contains specific codons recognized by E. coli. An example of such a variant is presented in example 8.

In this context, the term "gene" is used to denote a DNA sequence, which is included in the production of the polypeptide chain and which includes sections preceding and following the coding sections of 5'forward and 3'reverse sequence/ as well as terminating sequences, introns, which are located between individual coding segments, the exons or in the 5'-forward direction or the 3'backward areas. The plot in the 5'-forward direction contains a regulatory sequence, which contacts the expression of a gene, usually the promoter, 3'-phase in the opposite direction contains sequences that are involved in termination of transcription of the gene, and optionally the sequence, the responsibility is passed the present invention relates to expressing the system, containing the nucleotide sequence as previously described encoding the polypeptide of the present invention, the system that contains the 5'-flanking sequence capable of expression of the specified nucleotide sequence.

In particular, the present invention relates to the replicated expression vector that provides for expression of the nucleotide sequence of the present invention. A particular variant of the present invention relates to the replicated expression vector, denoted by pS 507, which was deposited on may 11, 1993, in the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the registration number DSM 8282 in accordance with the requirements of the Budapest agreement, and expression vectors that Express a nucleotide sequence that differ from the nucleotide sequence of the deposited specified vector expression, but which encode the same polypeptide or an analogue or variant, which have SCCE activity.

In other words, the scope of the invention also includes a replicable expression vector, as described earlier, in which the expressed nucleotide of posledovatelnosti fact, that at least one nucleotide has been deleted, replaced or modified, or at least one additional nucleotide has been incorporated so that the nucleotide sequence that encodes the polypeptide, would have SCCE activity.

Moreover, the present invention relates to a plasmid, designated pS 500, which was deposited on may 11, 1993, in the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the registration number DSM 8281 in accordance with the requirements of the Budapest agreement, and plasmids containing the nucleotide sequence that differs from the nucleotide sequences shown in sequence ID No. 1, but which encodes the polypeptide presented in sequence ID No. 2, or an analogue or variant of it, which have SCCE activity, or which hybridized with nucleotide sequence, presented in sequence ID No. 1, or part of it in the harsh conditions of hybridization.

In the scope of the present invention also includes non-human organism, which contains the expression system of the present invention. Organisms that can be used in this aspect of the invention, include the cells, derived from multicellular organisms, such as fungi, insect cells, plant cells, mammalian cells or cell lines. If the organism is a bacterium, preferably a bacterium belonged to the genus Escherichia, such as E. coli.

In addition, the present invention relates to a plasmid vector containing a DNA sequence that encodes the polypeptide of the present invention or the polypeptide of the merger, as defined here. In one of the most important variants of the DNA fragment, or analog, or suppositionally of the present invention or the DNA fragment merge of the present invention may contain replicated expression vector that can replicate in the host organism or cell line.

Such a vector can be, in particular, a plasmid, a phage, kosmidou, mini-chromosome or virus. In an interesting variant of the invention the vector may be a vector which, when introduced into the cell host is integrated into the cellular genome.

If you use a higher organism, to obtain polypeptides can be used transgenic technique. Examples of suitable animals are sheep, cattle, pig, what control tone-specific regulatory elements. The resulting protein can then be subjected to post-translational modifications to obtain the polypeptide of the present invention.

Transgenic mammals /this does not apply to the person/ of the present invention are due to the introduction of a "transgene in embryonic micheneau the selected cell of the animal. In one aspect of the invention, the transgene is a DNA sequence that is able to produce the desired phenotype when it is contained in the genome of the cells of the transgenic mammal /not human/. In specific embodiments, the transgene contains a DNA sequence encoding the polypeptide of the present invention, the transgene is capable of being expressed to produce the polypeptide.

The inclusion of expressing the system in the germ line /germline/ mammal can be performed using any suitable technique, such as described by Hogan et al., 1986, or in WO 93/04172.

In a particular aspect of the invention the nucleotide sequence of the invention may contain another nucleotide sequence encoding a polypeptide that is different from or identical to the polypeptide of the present invention, fused in frame with a nucleotide sequence in order to obtain the fused polypeptide, this polypeptide is another interesting aspect of the present invention, see example 8. If using recombinant DNA technology, fused DNA sequence can be incorporated into a suitable vector or genome. In another embodiment, one of the nucleotide sequence inserted into a vector or genome contains another nucleotide sequence. The polypeptide of the fusion can also be obtained by embedding the two nucleotide sequences separately and allowing the flow of expression. The host organism, which can be both eukaryotic and prokaryotic origin, grown in conditions which ensure the expression of the fused sequences. Then merged the purified polypeptide, and the polypeptide of the present invention is separated from his partner, merge, using suitable methods.

Thus, one aspect of the present invention relates to a method for producing a polypeptide of the present invention, which involves the following stages:

/a/ embed nucleotide sequence of the present invention in the expression vector,

/b/ transformation of a suitable host organism with the vector obtained at the stage of /a/,

/c/ ku

/d/ collecting polypeptide and

/e/ optional post-translational modification of the polypeptide.

In the scope of the present invention also includes the previously described method in which the obtained polypeptide emit method that includes one or more of the stages, similar to affinity chromatography using immobilized native or recombinant SCCE polypeptide or antibodies reactive with the specified polypeptide, and/or procedures chromatographic treatment and electrophoresis.

The polypeptide obtained as indicated previously, it is possible post-translational modification, for example, by heat treatment, chemical treatment /formaldehyde, glutaraldehyde and so on/ or enzymatic processing /peptidases, proteases and protein-modifying enzymes/. This polypeptide when produced in the body can be processional in other ways than those which occur in its production in the natural environment. For example, glycosylation is often achieved when the polypeptide is expressed in such a cell of a higher organism like yeast or preferably mammals. Usually glycosylation is associated with amino acid residues Asn, Ser, Thr or hydroxylysine. trevelyn the host organism.

After expression in accordance with the present invention the polypeptide in the organism or cell line, the polypeptide can be used as it is, or it can first be isolated from the organism or cell line. If the polypeptide expressed in the form of secreted product can be cleared directly. If the polypeptide expressed in the form of the associated product, you may need partial or complete destruction of the host before cleaning.

Examples of procedures used for the purification of polypeptides include:

/i/ immunoadhesin or affinity chromatography with antibodies

(ii) affinity chromatography with a suitable ligand,

/iii/ other chromatographic procedures such as gelfiltration, ion exchange or high-performance liquid chromatography, or modifications of the above

/iv/ electrophoretic procedures such as polyacrylamide gel electrophoresis, electrophoresis denatured polyacrylamide gel, agarose gel electrophoresis and isoelectric focusing,

/v/ any other specific techniques of solubilization and/or treatment.

The present invention relates also to almost pure SCCE on the CI does not contain other components, that is, other polypeptides or carbohydrates, which could be formed in the process of receiving and/or releasing polypeptide, or any other way to be together with the polypeptide. The degree of purity of the protein can be evaluated by electrophoresis on SDS gel as described in example 3.

The polypeptide can be identified according to the method of example 4 using SBTJ affinity chromatography or by using affinity chromatography on immobilized antibodies, e.g. antibodies TE4b, in a way known in the art. A high degree of purity of the polypeptide of the present invention can be advantageous if the polypeptide are going to use in pharmaceutical or cosmetic compositions. In addition, due to the high degree of purity, almost pure polypeptide can be used in smaller quantities than the polypeptide normal lower degree of purity for most purposes.

In one aspect of the present invention pure polypeptide can be obtained from a suitable cell line, which expresses the polypeptide of the present invention, as described in example 9. In addition, the polypeptide of the present invention can be well-known methods of liquid or termofosfatnoy. In another embodiment, the polypeptide can be synthesized by connecting the individual amino acids, forming fragments of the polypeptide sequence, which are then combined to obtain the target polypeptide. These methods, therefore, are the next interesting aspect of the invention.

A very important aspect of the invention relates to pharmaceutical compositions, cosmetic compositions or compositions for treating the skin, which contains a polypeptide having SCCE activity, and a pharmaceutically and/or cosmetically acceptable excipient. The composition may contain purified native protein or recombinant polypeptide of the present invention, or proenzyme or protein fusion - form of the polypeptide of the present invention, which can be activated through proteolytic cleavage.

Proenzyme the form of the polypeptides of the present invention can be classified as "Palekastro form", that is, the connection that after the subsequent proteolytic cleavage into an active form.

Especially, but not exclusively, the present invention relates to compositions suitable for surface coating, for example, application application, can be creams, ointments, lotions, liniments, gels, solutions, suspensions, pastes, patches, sprays, shampoos, Soaps, hair conditioners or powders.

Surface coating involves the application on or near the body parts that are considered pathological changes, for example, such external parts of the body, as the skin surface. The application may just be the distribution of the composition on the surface or may include any device designed to strengthen and consolidate the contact of the composition and the pathological lesions, for example, the closing of the bandage, such as an insulating adhesive tapes coated with the composition of the present invention. These compositions may be impregnated or can be distributed on the swabs, plasters, bandages, gauze, sponge materials, pieces of cotton, etc., In some cases, you can use the composition in the form of injections directly into the affected area or close to it.

The composition for surface coating of the present invention may contain 1-80% of the active compound by weight calculated on the total weight of the preparations, for example, 0.001 to 25% weight/weight of active compounds, for example, 0,1-10%, 0,5-5% or 2-5%. Part of cosily, containing SCCE, Pro - SCCE or inhibitor of SCCE in combination with other pharmaceutical and/or cosmetic compounds.

The composition of the present invention is usually applied 1-10 times a day, depending on the type, extent and location of the affected area.

For the surface coating of the drug can be prepared according to conventional pharmaceutical practices and pharmaceutical excipients that are commonly used for surface coating. The nature of the medium, which is used for preparation of each specific composition depends upon the method of application of the composition. In addition to water can be used in the compositions and other media, which include solids or liquids, as a softening means, solvents, humectants, thickeners and powders. Examples of this type of media that can be used either individually or in mixtures, consisting of more than one carrier, are the following:

Sleek tools: stearyl alcohol, monoricinoleate glycerol, glycerol monostearate, propane-1,2-diol, butane-1,3-diol, cetyl alcohol, isopropyltoluene, stearic acid, isobutylacetate, isolatedstore, aleinov ilpolitical, di-n-BUTYLCARBAMATE, isopropylmyristate, isopropyl, isobutylester, butilstearat, polyethylene glycol, triethylene glycol, lanolin, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, Boutilimit, ezoterikova acid, palmitic acid, isopropylidene, laurinlactam, myristylated, decillia, myristoleate.

Solvents: water, methylene chloride, isopropanol, castor oil, monotropy ether of ethylene glycol, monobutyl ether of diethylene glycol, monotropy ether of diethylene glycol, dimethyl sulfoxide, tetrahydrofuran, plant and animal oil, glycerin, ethanol, propanol, propylene glycol and other glycols or alcohols, fixed oil.

These moisturizing agents like glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin.

Such powders such as chalk, talc, kaolin, starch and its derivatives, resins, colloidal silicon dioxide, sodium polyacrylate, chemically modified magnesium aluminum silicate, hydrated aluminum silicate, carboxyvinyl polymer, sodium carboxymethyl cellulose, glycol monostearate.

Such gelling or the way the oz, the carboxymethyl cellulose or oxidized cellulose, cellulose resin, guargum, resin acacia, karaya and tragakant, bentonite, agar-agar, alginates, carbomer, gelatin, bladderwrack, ceratonia, dextran and its derivatives, gum ghatti, hectorite, resin xantam, ispaghula husk.

Such polymers like polyacrylic acid or polymers polyglycolic acid, or their copolymers, paraffin, polyethylene, polyethylene oxide, polyethylene glycol, polypropyleneglycol, polyvinylpyrrolidone.

Such surface-active agents are nonionic surface-active agents, for example, glycol and esters of glycerin, esters and ethers of macrogol, ethers and esters of sugars, such as sorbitane esters, such ionic surface-active agents, as aminoil, metallic Soaps, sulfated fatty alcohols, alkylarylsulfonate, sulfated oils and impolitically surface-active agents and the lecithins.

Such buferiruemoi agents, as salts of sodium, potassium, aluminum, magnesium or calcium /such as chlorides, carbonates, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tartratami/.

For the surface coating of the pH of the composition, in principle, can vary in a very wide which corresponds to the proteolytic activity of the polypeptide, for example, a pH of from about 4 to 8. Mentioned previously buferiruemoi agents can be used to establish the desired pH.

The preparations of the present invention may also contain other additives such as stabilizing agents, preservatives, agents that facilitate dissolution, coloring agents, chelating agents, gel-forming agents, bases ointments, pH regulators, antioxidants, perfumes and agents that protect the skin, and so on, If the composition is prepared in the form of soap or shampoo, it can, furthermore, contain foaming agents, colouring agents and/or conditioners.

Typical preservatives include parabens, formaldehyde, Kathon CG, Bronidox, Bronopol, perchlor-meta-cresol, chlorhexidine, benzylaniline etc.

If the composition of the present invention are in the form of shampoo or soap, you can use basic ingredients, and the typical basic soap and shampoo include components such as betaine, nutriceuticals, nonilfenol, imidazole, sulfosuccinate, degreasing agents, moisturizers and conditioners.

Moreover, it may be advantageous to create drugs with modified allocation, in which the active compound is included in a polymer matrix, or in limer.

The composition can be obtained in accordance with conventional pharmaceutical practice, and they can be:

Semi-solid compositions: gels, pastes, compounds.

Liquid compositions: solutions, suspensions, emulsions.

As mentioned, the pharmaceutical composition of the present invention may contain the polypeptide of the present invention, or its functional derivative, or a combination of such compounds. Examples of suitable functional derivatives include pharmaceutically acceptable salts, especially the salts, which are suitable for use on the skin. Examples include pharmaceutically acceptable salts aminopentyl, for example, salts of acids, which give the anions, which are pharmaceutically acceptable, especially for use on the skin. Examples include phosphates, sulfates, nitrates, iodides, bromides, chlorides, borates, and anions derived from carboxylic acids, which include the acetates, benzoate, stearates, etc.

Other derivatives aminopentyl include amides, imides, urea, carbamates, etc.

Other suitable derivatives include derivatives of carboxylic groups of the polypeptide of the present invention, including salts, esters and amides. PR, the Inca, aluminium, iron /2 and 3/, ammonium and lower /C1-6/-alkylamine. Esters include lower alkylsilane ethers.

Examples of compositions in example 11 illustrate examples of pharmaceutical and cosmetic compositions and compositions for skin care in accordance with the present invention, but they in no way limit the scope of the compositions of the present invention.

It is shown that the cosmetic compositions for skin care containing the native or recombinant SCCE, active acne, xeroderma or other hyperkeratotic conditions such as corns and keratosis pilaris.

There are different stages of acne vulgaris. Consider that it is desirable to introduce SCCE on those stages, when there is a common keratinization in the ducts of the sebaceous glands, which leads to the formation of blackheads and clogged duct, so it may be advantageous to introduce a substance that inhibits SCCE on stage when inflammatory acne lesions are the predominant feature.

Thus, one aspect of the present invention refers to the use of the polypeptide for the treatment or prevention of acne, xeroderma and other hyperkeratinized States, so rmaceuticals composition, containing the native or recombinant SCCE suitable for the treatment or prevention of various ichthyosis, acne, psoriasis or other inflammatory skin disorders such as eczema with hyperkeratosis, microbial infection, and for healing, especially when surface applied.

Another aspect of the invention relates to the use of the polypeptide having SCCE activity, for the preparation of pharmaceutical compositions for the treatment or prevention of various ichthyosis, acne, psoriasis or other inflammatory skin diseases accompanied by hyperkeratosis, such as eczema.

Another aspect of the invention relates to a method of treatment and/or prevention of various ichthyosis, acne, psoriasis and other inflammatory skin diseases accompanied by hyperkeratosis, such as eczema, and the method includes the introduction of the required treatment to the patient a therapeutically or prophylactically effective amount of the polypeptide having SCCE activity. The treatment can be prophylactic, palliative or curative.

It is assumed that the "cascade system" proteoliticheskikh enzymes exist in the skin environment, which is similar to the system aktiverat, the SCCE activity can inhibit using "SCCE inhibitor".

In the case of certain skin diseases, such as autoimmune diseases-water or acantholycosa diseases, for example, family disease or illness Darir's, there is a cohesion between keratinocytes and corneal life epidermal layer /see impairment of cell cohesion in the living epidermis/. Believe that this process is carried out by proteases, and therefore it can be affected by introducing a compound that could inhibit the enzymatic activity of native SCCE. It may also be advantageous to introduce SCCE inhibitor for the treatment of psoriasis and other inflammatory skin diseases in conditions where inflammatory component is dominant.

In another aspect, the present invention relates to the use of SCCE inhibitor that exerts an inhibitory effect on the enzymatic activity of native SCCE, for the preparation of pharmaceutical compositions for the treatment or prevention of autoimmune diseases) or acantholytic diseases, such as family disease or illness Darier's.

In this context, the term "SCCE inhibitot polypeptide sequence or suppositionally invention or similar so his SCCE activity is declining. This reduction can be defined, for example, carrying out the experiment in accordance with the scheme of example 3.2 using potential SCCE inhibitor as an inhibitor. These compounds may be organic molecules, small peptides or large polypeptides or derivatives of any of the previously mentioned. This approach may find application in the program of screening drugs to identify SCCE inhibitors.

Thus, the following variant of the present invention relates to a method of identifying a compound that affects the enzymatic activity of native SCCE, including the use of recombinant polypeptide of the present invention.

In particular, the present invention relates to a method of identifying a compound that has inhibitory effect on the enzymatic activity of native SCCE.

In another aspect, the present invention relates to a method of identifying compounds that enhance the enzymatic activity of native or recombinant SCCE.

An important application of the recombinant polypeptides of the present invention is a screening E. the invention relates also to a method of identifying compounds which is able to turn proenzyme form SCCE in active SCCE containing the polypeptide of the present invention.

The concept of the present invention also includes the use of amino acid sequence, as described previously, to determine the three-dimensional structure SCCE polypeptide for use in creating the substance capable of binding SCCE polypeptide, particularly for use in the creation of a medicinal substance that binds to the active site of the enzyme.

Finally, an important aspect of the present invention are different ways of regulation of activity demonstrated SCCE a polypeptide. This activity may be important for a variety of painful conditions, as mentioned previously.

DNA or RNA fragment complementary to at least part of an mRNA corresponding to the polypeptide of the present invention or its equivalent, may be effective in stopping broadcast SCCE vHYR in human cells, and thereby, inhibition of synthesis of the polypeptide /polypeptide/. This approach, which might be interesting for painful conditions that are experiencing higher than normal expression of SCCE, such as autoimmune disease-Esten as the antisense oligotherapy and therefore, the present invention also includes this approach.

Description of drawings

Fig. 1

Unipolar cell, otshelushivshihsya with the corneal layer of the heel surface in vitro.

The surface of the fabric, which in vivo looked up, Fig. 1 is the surface down. It should be noted that there has been a progressive cellular dissociation on this surface in the process of incubation, and on other surfaces of cell dissociation is not observed.

Fig. 2

The time dependence and the effect of Aprotinin on the selection of cells from the corneal layer of the heel surface, incubated without /mugs/ and /triangles/ Aprotinin. Are average /total values for the four samples and the interval of confidence.

Fig. 3

Anti-desmoglein /anti-DG1/ reactive components in solid corneal layer of the heel surface and dissociated cells.

A. Painted Coomassie blue SDS-PAGE.

C. Immunoblot. 1-3: knitted fabrics, undiluted /1/ diluted 1/3 /2/ diluted 1/9 /3/. 4-5: dissociatively cells, undiluted /4/ diluted 1/3 /5/.

Marked only apparently intact DG 1 SIG. 4

The time dependence and the effect of Aprotinin on the degradation desmoglein 1 /DG 1/ in the corneal layer of the heel surface, undergoes exfoliation of cells in vitro. Densitometric scanning of immunoblots extracts the corneal layer of the heel surface, incubated in the absence (A) and in the presence of /B/ Aprotinin /15 μm/. The peak at 160 KD corresponds to the intact DG 1. Peaks at 95 and 80 KD correspond to degradation products of the protein (see Fig. 3/. Notes effective inhibition by Aprotinin degradation DG1.

Fig. 5.

The influence of zinc ions /A/, hemostatis and leupeptin /B/ on the degradation desmoglein 1 /DG 1/ in the cells of the corneal layer of the heel surface, exfoliate in vitro. Marked inhibition of the transformation of the anti - DG1 positive components of the 160 KD 95 and 80 KD due to zinc ions and hemostatis, but not at the expense leupeptin.

Fig. 6.

The activity of the hydrolysis of peptides associated with cells of the corneal layer of the heel surface. For the hydrolysis of the two substrates observed by measuring the change in absorption at 405 nm. The average for the three inkubirovanii. Squares = S-2586; circles = S-2288.

Fig. 7

the pH-dependence of corneocyte-related S-2586 hydrolysing Cl.

Fig. 8

Zymography showing caseinolytic activity in extracts of dissociated cells of the corneal layer of the heel surface. Cm. the text of example 2.2 for details of the experiment.

A: Comparison of the enzyme from the heel of corneocytes extracted samples buffer Laemmli without reducing agent /Sc/S/ and KCl /Sc/k/ with bovine chymotrypsin, a 0.125 ng /s/ and bovine trypsin, 0.5 ng /t/. Before electrophoresis KCl-extract cialiswhat against 5 mm Tris-HCl, pH 6,8, and SDS and glycerin is added to the final concentrations as in the exemplary buffer; 10 μl added to all bands. The molecular weight markers are shown on the left.

B: Dependence on pH of the incubation buffer. The enzyme source = D - extracts dissociated heel of corneocytes. Buffers for pre-treatment Triton X= 100 and incubation: 0.1 M sodium acetate /pH 4.0 and pH 5.5, 0.1 M ris-HCl /pH 7.0 and pH 8.0/. Other conditions as for A.

C: Effect of inhibitors. SC = SDS - extract the heel of corneocytes; C = bovine chymotrypsin; t - bovine trypsin. Inhibitors are present during the pre-treatment Triton X-100 and subsequent incubation. The final concentration leupeptin is 160 μm, 15 μm Aprotinin, hemostasia 40 μm and an ion is a Buffer for pre-treatment and incubation = 0.1 M Tris-HCl, pH 8 at a final concentration of DMSO 1% /vol/vol/. Other conditions as in A.

Fig. 9

Comparison of SCCE, bovine chymotrypsin and cathepsin G of human rights in respect of the action of inhibitors /A; Aprotinin; B; hemostatic, C; zinc sulfate/ substrate specificity and /D/. For A-C the enzyme activity without inhibitor taken for 100%. B On the activity of the enzyme with MeO-Suc-Arg-Pro-Tyr-pNA are for 1 arbitrary unit.

Fig. 10.

Affinity chromatography on covalently linked inhibitor of trypsin soybean/SBTJ. Dotted line: absorption at 280 nm reflects the concentration of protein in the eluate. The solid line with empty squares: the activity of the hydrolysis of peptide with S-2586 as a substrate, is shown as a change in absorption at 405 nm. The solid line with empty circles: the Activity of the hydrolysis of peptide with S-2288 as a substrate, is shown as a change in absorption at 405 nm, multiplied by 10.

Fig. 11.

SDS-PAGE and Coomassie-blue staining of /A/ and zymography after SDS-PAGE with copolymerizable casein /B/ factions 2,6 and 10 from the chromatograms shown in Fig. 10. The molecular weight markers are presented on the left. B: 1: a group caseinolytic components that can inhibit leupeptin /160 mcm/. C: ptx2">

SDS-PAGE SCCE, purified using affinity chromatography on SBTJ-Affigel 15/ 12.5% gel.

1: unrecovered sample.

2: Recovered sample.

The molecular weight markers are presented on the left.

Fig. 13.

The N-terminal amino acid sequence of SCCE.

Asterisks indicate uncertainty for prospective cysteines in positions 7 and 9. Question marks indicate the position in which it is impossible to determine amino acid derivatives, but in the next stages, there is no reduction in output.

Fig. 14.

Characterization of monoclonal antibodies TE4b and TE9b by immunoassay and Western blot turns.

a: Coomassie - stained with 12.5% SDS-PAGE, non conditions.

Lane 1: molecular weight Markers.

Lane 2: KCl - extract prepared by the method of example 3.1, dissociated heel of corneocytes.

Band 3: SCCE, purified by the method of example 4.1 using affinity chromatography on made insoluble soy trypsinogen the inhibitor.

b: zymography of immunosurgery 12.5% SDS-PAGE c 0,1% copolymerizable thermodenaturation casein, Coomassie - stained gel.

Lane 1: Markers is penditures immunoassay with /left to right /oab TE4b /20 mcg/, oab TE9b /10 µg/, oab PZ /10 µg/ and superyoung phosphate saline. Moab PZ is a mouse monoclonal antibody Jg-Gl - Carr type to the protein zone of pregnancy, and was used as an unrelated negativy control.

C: Immunoblot of a 12.5% SDS-PAGE /non-conditions/.

Lane 1: biotinylating the molecular weight markers, detected by the conjugates of alkaline phosphatase-avidin /BioRad/. Electrophoretic processed sample in the bands 2, 4 and 6 is the same as in lane 2 in a, and in lanes 3, 5 and 7 is the same as in lane 3 in a.

Lanes 2 and 3: the First antibody = oab TE4b, 0,2 μg per ml.

Lanes 4 and 5: the First antibody = oab TE9b, 0.1 mg/ml

Lanes 6 and 7: First antibody = oab PZ, 0.1 mg/ml

The edge of arrows in a-c indicate the relative molecular weight /top-down/ 93, 66, 45, 31, 22 and 14 KD, respectively.

Fig. 15.

Fig. 15 shows the plasmid pS 500. This plasmid contains the full length cDNA SCCE human cloned pS C19. For more information, see example 6.

Fig. 16.

Northernblues with mRNA derived from human epidermis. Poly-T-purified RNA corresponding to approximately 100 g of total RNA applied to each band.

1: the lead with probe, obtained from 655 bp Hinc 2/Bgl 2 fragment SCCE-cDNA.

Fig. 17.

a. Coomassie - stained SDS-PAGE, 12.5% gel. 1 and 2: PBS-Triton X-100 insoluble residue treated with ultrasound TG 2 cells transformed pS 510 and pS 511, respectively, and induced by iPTG. 3: SCCE isolated from the corneal layer of the heel surface of the person.

b. Immunoblot with chicken pre-immune serum /1-3/ and chicken anti - SCCE /4-6/ 1 & 4: TG 2 cells transformed pS 510 and induced by IPTG 2 and 5: TG 2-cells, transformed pS 511 and induced by IPTG. 3 and 6: SCCE isolated from the corneal layer of the heel surface of the person.

Samples are prepared by boiling in sample buffer with mercaptoethanol.

Fig. 18.

In Fig. 18 presents a circular map of the expression vector pS 507 constructed according to the method of example 9. Due to vector pS 507, is the expression of recombinant SCCE person in mammalian cells.

Fig. 19.

In Fig. 19 presents the analysis of expression of recombinant human SCCE gene pS 507 in mammalian cells.

Lane 1: RNA C127 cells.

Lane 2: RNA from the selected clone, 1:24, C127 cells, transfected with pS 507.

Lane 3: RNA from an expression vector, pS 147, which is identical to pS 507, except that it lacks SCCE cDNA sequence. The marker sizes are indicated on the left.

Fig. 20.

SDS-PAGE with subsequent Western blot turns SCCE, expressed in C127 cells.

Lanes 1 and 6: Pre-stained molecular weight markers /Bio-Rad, 106, 80, 49,5, 32,5, 27,5 and 18.5 kDa/.

Lane 2: 507 pS/C127, blend, T-flask.

Band 3: 507 pS/C127, blend, roller A.

Band 4: 507 pS/C127, blend, roller B.

Lane 5: Negative control pS 522/C127.

Band 7: Native SCCE obtained from the corneal layer.

Band 8: 507 pS/C127, clone 24, T-flask.

Band 9: 507 pS/C127, clone 24, the roller A.

Band 10: 507 pS/C127, clone 24, the roller B.

Fig. 21.

SDS-PAGE (a) and Western blot turns /B/ recombinant SCCE isolated from C127 cell culture medium by cells carrying plasmid pS 507.

Lanes 1 and 5: Pre-painted markers molecular weights /Bio-Rad, 106, 80, 49,5, 32,5, 57,5 and 18.5 kDa/.

Lane 2: Cell environment before cleaning.

Band 3: Unbound material collected from the chromatograms.

Lane 4: the Linked material, suirvey buffer with

low pH.

Lanes 1 and 10: molecular Markers of scales /Pharmacia 14-94 kDa/.

Lane 2: Native SCCE.

Band 3: Native SCCE.

Lanes 4-6: Recombinant SCCE, split, within 1 hour, 3 hours and overnight, respectively /460 ng/cell/.

Band 7: Trypsin in the same amount as in the samples in lanes 4-6, but without APMSF.

Band 8: Trypsin in the same amount as in the samples in lanes 4-6, in the presence of the same amount APMSF, and samples.

Fig. 23

The immunoblot N-glycosidase Ftreated native and recombinant SCCE. The samples are divided into 8-18% SDS-PAGE, and Western blot turns carried out as described previously.

Lane 1: molecular weight Marker. Molecular weight, counting from the: 106, 80, 49,5, 32,5, 27,5 and 18.5 kDa.

Lanes 2 and 3: recombinant SCCE, 0.3 and 3 μg, respectively.

Samples in lanes 3 and 5 treated with N-glycosidase F.

Examples

Example 1

Evidence that cells exfoliate from the surface of keratinized surface layer of the skin, contain decayed desmosomal proteins, and the enzyme, which seems to be responsible for this, is chymotrypsinogen proteinase, which was to ascertain the nature of the mechanism, responsible for cellular cohesion and dissociation of surface cells /desquamation/ the cornified layer of skin, Stratum corneum. Scales corneal layer thickness of 0.3 - 0.6 mm is cut parallel to the skin surface with the heel surface volunteers with normal skin. This cloth is dipped in superyoung phosphate saline with 0.1% nutriasia for three hours at room temperature and weakly attached cells with outer surface of /in vivo/ soskrebajut. Pieces 1 mm thick, cut perpendicular to the surface of the fabric, then placed in a medium containing 0.1 M Tris-HCl pH 8, 5 mm EDTA, 0,1% sodium azide and 0.45% agarose, just before gelation environment due to the presence of agarose. After incubation for 0.5 and 15 hours at 37oC, the gel pieces with fabric frozen on dry ice. 20 μm frozen slices cut perpendicular to the skin surface in the cryostat, strengthen and examined under phase contrast microscope. There is a continuous unipolar exfoliation of cells in slices of the corneal layer of the heel surface, inkubiruemykh in vitro /in Fig. 1/. Cells are sloughed off only on the surface of the tissue, which in vivo were outer. Thus, the observed percent of the developed method for quantitative determination exfoliate, which allows to investigate the influence of different parameters, such as temperature, pH and enzyme inhibitors. Cylinders corneal shell heel surface with a diameter of 3 mm was obtained by using the device for biopsy of scales tissue taken and frozen weakly attached surface cells, as mentioned earlier in 1.1. These cylinders /with a specific surface area, which in vivo facing out/ incubated in 0.5 ml of medium containing 0.1 M Tris-HCl pH 8, 5 mm EDTA, 0,1% sodium azide and Aprotinin /410-6mol/l/ or without /Bochringer-Mannheim, Germany/ 1.5 ml flasks Eppendorf at 37oC for 5, 10 and 20 hours, and then stirred for 10 seconds in a Vortex mixer for the Department of dissociated surface cells. The remaining tissue is removed and placed in fresh medium for further incubation. Ampoules with separated cells centrifuged for 2 minutes at 5000 g to collect the cells. Cellular precipitation is washed once with 0.5 ml superyoung phosphate saline solution, and then treated at 60oC for 1.5 hours 0.6 ml of sodium hydroxide. Alkali-soluble protein determined according to Lowry et al., 1951, and take as a measure of the amount of separated cells. Poluchennyh inhibitors /Aprotinin, inhibitor soybean trypsin, pepstatin /Bochringer-Mannheim, Germany/ and iodoacetamide /Sigma, St. Luis, MO/. Individual cylinders of tissue 2 mm prepared and incubated with the medium with various potential inhibitors /or without/ at the concentrations indicated in table 1 below, for 16 hours at 38oC, and the separated cells counted as described previously. It should be noted that EDTA /which is a metalloproteinase inhibitor was included in the incubation environment for optimal speeds rejection of the cells.

It was found that inhibitors semipretioase, Aprotinin and inhibitor soybean trypsin effectively inhibit peeling of cells /Fig. 2 and table 1/. As these two substances were inhibitors, but inhibitors of metalloproteinases /EDTA/, tiaprofenic /iodoacetamide/ and aspartic proteases /pepstatin/ were not, it was concluded that semipretioase participates in the observed process. Have also come to the conclusion that cell cohesion in the corneal layer depends on protein structures, and that a mechanism similar to the observed in vitro, should also work in the process of desquamation in vivo / Lundstrom and Egelrud, 1988/.

In further studies, letocnazelre into two separate stages. The first stage takes place regardless of whether EDTA in the incubation media or not. The second stage occurs only in the presence of EDTA. The first stage can inhibit homestation and zinc ions, in addition to Aprotinin. The second stage can be inhibition by Aprotinin and homestation /Lundstrom and Egelrud, 1990 a/. Hemostatis is a small molecule inhibitor of proteinases with chymotrypsin-like specificity to substrates. In addition, it was found /Lundstrom and Egelrud, 1990 a/ that leupeptin, a small molecule inhibitor of proteinases with trypsin-like specificity to the substrate, does not affect in vitro exfoliation of cells.

Most likely that protein structures, which are responsible for cell cohesion in the corneal layer, and thus are potential candidates for degradation in the previously described desquamation cell peeling, are desmosomes. Desmosome consist of two symmetrical halves, which are located in contiguous cells. The two halves are connected in the extracellular space transmembrane proteins, called desmogleins.

1.3 the Fate desmoglein 1 /DG1/ in the process of desquamation of cells in vitro

ISS is STI. The corneal layer of the heel surface is incubated, as indicated earlier, 1.1, and still cohesive fabric is separated from the dissociated cells. Cells and tissue extracted in buffer containing 0.1 M Tris-HCl pH 9, 9 M urea, 2% dodecyl sulfate, 1% mercaptoethanol, 1 ml of the buffer to 20 mg tissue, for 15 hours at 37oC. Extracts were prepared for electrophoresis on polyacrylamide gel in 7.5% gels in the presence of nitrilotriacetate /SDS-PAGE/ in accordance with the instructions of Laemmli /Laemmli, 1970/, followed by electrophoretic transfer /Towbin et al., 1979/ on nitrocellulose membrane /Bio-Rad, Richmond, CA), which was probed rabbit polyclonal anticorodal obtained against DG1 allocated bullish Mord /Gorbsky et al., 1985/. The bound antibodies detected using alkaline phosphatase conjugated goat anti-rabbit immunoglobulins /Dio-Rad, Richmond, CA/, /lake, et al., 1984/.

The results obtained are shown in Fig. 3. The amount of sample added to immunoblot, set this to install approximately equal concentrations of protein (which can be determined visually by stained Coomassie blue SDS-PAGE gels) for solid tissues and dissociated cells. Spend several dilutions extraintf in whole corneal layer and dissociated cells.

If still solid fabric and dissociatively cells are extracted separately, it is found that while still solid fabric contains only apparently intact DG1, dissociatively surface cells contain only the expected decomposition products of protein /Fig. 3/.

In Fig. 4 and 5 presents the impact of Aprotinin, zinc ions, hemostasia /Bochringer-Mannheim, Germany/ and leupeptin /Bochringer-Mannheim, Germany/ on the degradation of DG1 in the process of in vitro cell exfoliation with corneal layer of the heel surface.

First, investigated the time dependence and the effect of Aprotinin on the degradation desmoglein 1 /DG1/ peeling cells of the corneal layer of the heel surface nor vitro. Extracts of the corneal layer of the heel surface is incubated, as indicated in 1.2 /but without allocation of solid tissue from dissociated cells before extraction of/ in the presence of Aprotinin /15 ál/ or without him, and then extracted after 0, 6, 12 or 24 hours. SDS-PAGE and Western blot turns performed as described previously. Densitometric scanning of immunoblots spend on Shimadgu CS-900 scanning device with a flying spot /Shimadgu, Kyoto, Japan/ in reflected light at 560 nm in a zigzag mode. The obtained result is ledout the influence of zinc ions, hemostasia and leupeptin on the degradation desmoglein 1 /DG1/ in the process exfoliate the corneal layer of the heel surface in vitro. Scheme of the experiment is similar to that described previously. Incubation lead within 24 hours with zinc sulfate at concentrations of 0, 1 or 5 mm, and homestation or leupeptin in concentrations of 330 μm. The results obtained demonstrate the inhibition of the transformation of the anti-DG1 positive components of the 160 KD and 95 KD due to zinc ions and hemostatis, but not at the expense leupeptin.

From these results, it is evident, that should Aprotinin, zinc ions and hemostatis were inhibitors and leupeptin not. Thus, the nature of the inhibition of the degradation of DG1 was the same as to exfoliate the corneal layer of the heel surface in vitro /Lundstrom and Egelrud, 1990 b/.

It was considered important to demonstrate that mechanisms similar to those responsible for exfoliation of cells from the corneal layer of the palms soles, are also manifested in the corneal layer of skin from other parts of the body, rather than taken from the palms and soles. Takahashi et al. / Takahashi et al., 1987/ reported that a mixture of detergents N,N-dimethyldodecylamine /Sigma, St. Luis, MO/ and nitrilotriacetate /Bio-Rad, Richmond, CA/ as molar ratio is nom by trypsinization whole epidermis. To avoid contamination due to exogenous trypsin, biopsy normal human skin with gluteal sections incubated at pH 8 with the above mixture of detergents, EDTA /Egelrud and Lundstrom, 1990/. It was found that under such conditions the keratinized layers dissociated to single cells. Adding Aprotinin to the incubation environment prevents this cell dissociation. It was concluded that cellular cohesion and corneal layers, not taken from the palms soles, depends on the structures of tissues, that is, desquamation in such tissue-dependent proteolysis, and that this tissue contains a proteinase that can catalyze such proteolysis. As cell dissociation includes only the corneal layer, and not the deeper layers, not neurogenesis layers of the epidermis, it was concluded that responsible for this process proteinase is in the deeper layers in the inactive or inhibited state. Example 2

Opening chymotrypsinlike enzyme corneal layer /SCCE/: proteases, which meets the criteria responsible for the degradation of intercellular cohesive structures in the corneal layer in vitro and possibly also in vivo.

From the experiments of example 1 was concluded that the proteinase, OA heel surface, must have the following properties:

1. It must be present in the corneal layer.

2. It must be the serine proteinase.

3. It must have chymotrypsin-like specificity to the substrate and the nature of inhibition similar to that observed when peeling cells in vitro and is associated with the degradation desmoglein 1.

4. It must be localized extracellularly in the corneal layer.

5. It should be dependent on pH, which would allow her to be active under physiological conditions, as the pH of the corneal layer is about 4.5 to 6.

6. As exfoliation of cells from the corneal layer of the heel surface in vitro flows continuously during prolonged incubation time, even if the volume of the incubation medium is very large compared with the volume inkubiruemykh pieces of fabric, or if the incubation environment re changes in the incubation process, it seems reasonable to assume that the responsible enzyme is associated with the tissue so that it is not possible to extract it in the incubation medium during incubation.

The following two experiments led to the discovery of SCCE /Egelrud and Lundstrom, dissociatively cells of the corneal layer of the heel surface /corneocytes/ produced by incubation of the corneal layer of the heel surface according to the method of example 1. Cells filtered through nylon mesh hole size of 10 μm, and then washed three times with ten volumes of 0.1 M Tris-HCl pH 8, 5 mm EDTA and three times with 0.1 M Tris-HCl pH 8. These cells then incubated with two types of chromogenic proteinase substrate S-2288 or S-2586 /Kabai Diagnostica, Stockholm, Sweden/:

Ile-Pro-Arg-p-nitroanilide /S-2288/ split wide range of serine proteases with arginine-specificity /for example, trypsin/. Arg-Pro-Tyr-p-nitroanilide /S-2286/ is a substrate for chymotrypsin-like proteinases.

In the full volume of 120 ál of each reaction mixture contains a 0.07 M Tris-HCl pH 8, 0.1% of sodium azide, 1, 2, 5, 5 or 10 μl of a 25% suspension of washed corneocytes with the heel surface and 1.04 mm /S-2586/ or 1.25 mm /S-2288/ substrate. After incubation for 5 hours at 37oC in microtiter plates reaction is stopped by adding 125 ál of 10% acetic acid. The cells allowed to settle, and 200 μg of each supernatant transferred into a new cell. For the hydrolysis of the two substrates is monitored by measuring the change in absorption at 405 nm, after the cells are transferred into a Behring Elisa Processor /Behruigwerke, Marburg, Germany/.

As shown in Fig. 6, there is an enzymatic activity associated with disocyanate corneocytes heel surface, motosamokaty hydrolytic activity of S-2586 explore after that. Scheme of the experiment corresponds to the one presented previously, use 10 ál of a 25% suspension of washed corneocytes heel surface and buffers with different pH /sodium acetate, sodium phosphate or Tris-HCl/. The final concentration of the buffer is 0,07 M the results Obtained are shown in Fig. 7, from which it follows that the optimal activity at pH 7 to 8, but big enough and at a pH of 5.5.

In a further experiment examined the influence of EDTA, metal ions and inhibitors of proteases on the hydrolysis of S-2586 at pH 8 by proteases associated with suspendirovanie cells of the corneal layer of the heel surface. The experimental design presented earlier in table 2.

Notes to the table. 2:

a/ the Substance is present in the analytical mix.

b/ 25% suspension of cells of the corneal layer of the heel surface, prepared as described in the text was pre-incubated for 1 hour at room temperature in the presence of 1 mm PMSF /Sigma, St. Louis, MO/, dissolved in 2-propanol /final concentration of 2-propanol = 4% volume/volume/. Control suspension pre-incubated with only 4% of 2-propanol.

c/ Inhibitor, dissolved in dimethyl sulfoxide /DHS/. All environments, including controlfor serine proteases/, Aprotinin, an inhibitor soybean trypsin, hemostatis /inhibitor of chymotrypsin/, and zinc ions, but not leupeptin /trypsin inhibitor/ inhibit S-2586 hydrolytic activity. The nature of the inhibitor is therefore very similar to the character observed when peeling cells in vitro and is associated degradation desmoglein 1.

2.2 Zymography dissociated corneal layer of the heel surface

It was found that the enzyme responsible for the hydrolytic activity of S-2586 disclosed in 2.1, can be solubilisation if the corneocytes extracted with 1 M KCl in 0.1 M Tris-HCl pH 8. Therefore, experiments were carried out with demografia. For this reason, KCl extracts of corneocytes were prepared for electrophoresis by the method Lemli /Laemmli, 1970/, but without the reducing agent, in an exemplary buffer and not heating up the samples. Prepared samples by extraction dissociated corneocytes heel surface with an exemplary buffer Laemmli without a reducing agent at room temperature. For demografie adapted a modified procedure Horie et al. /Horie et al., 1984/. Electrophoresis on polyacrylamide gel are in the presence of nitrilotriacetate /SDS-PAGE/ by way Laemmli 12.5% gels with 1% copolymere is 0 for 1 hour at room temperature to remove SDS, and then incubated at 37oC for 15 hours. Then the gels stained Cocmassie-blue. Separate caseinolytic enzymes appeared as clear bands on a blue background. Cm. also the explanation of Fig. 8 for details of the experiment.

The results obtained are shown in Fig. 8. Extracts of corneocytes heel surface contain one main caseinolytic enzyme with an apparent molecular weight of approximately 25 KD. There are also smaller caseinolytic enzymes with molecular weights of about 30 KD. /These smaller components in Fig. 8 can not be seen clearly. It was later discovered that they can inhibit leupeptin, not homestation/. 25 KD enzyme has significant activity at pH 5.5 - 8. It is possible to inhibit artinya, zinc ions and homestation, but not leupeptin. Thus, he has the same nature of the inhibition, as indicated previously hydrolytic activity of S-2586. In experiments with gelchromotography exception /not shown/ 25 KD caseinolytic enzyme, as it was discovered, is shown together with S-2586 hydrolytic activity.

In subsequent experiments /not present/ with the previously described method in 2.2, it was found that the corneal layer is not with the palm is eocyte heel surface, from that point on will be called chymotrypsinogen enzyme corneal layer /SCCE/ /Lundstrom and Egelrud, 1991/.

It was possible also to obtain evidence that SCCE associated with corneocytes heel surface in such a way that it allows him to be active in the extracellular matrix of the corneal layer. This was done first demonstration that dissociatively the stratum corneum permeability to horseradish peroxidase /Mg 44 KD/. It was then shown that fibrinogen person /Mg 340 CD/ may degrade under the effect of the suspension of corneocytes, and that this degradation is possible to inhibit the same inhibitors that and SCCE. Hence we can conclude that the degradation of fibrinogen is associated with solubilizing enzyme /Egelrud, 1992/.

Example 3

Partial purification of chymotrypsinlike enzyme corneal shell /SCCE/ and analysis of proteases with chromogenic substrates

3.1 Obtaining KCl extracts of corneocytes heel surface

Getting dissociated corneocytes heel surface according to the method of example 1 was carried out on a larger scale, and were obtained KCl-extracts washed corneocytes heel surface containing SCCE, as described in example 2.

Getting the KCl EXT CNY layer heel surface is collected with the help of the Swedish society Pedicyrists.

Only used a material obtained using scissors or forceps. The material is not taken from his feet, affected by diseases associated with the peeling. Before sending the corneal layer was air-dried and Packed in plastic bags. In the laboratory the material was stored at -20oC until use.

For each subsequent stage affinity chromatography collect the extracts 1 and 2 (two receiving 50 g each) of the corneal layer of the heel surface

3.2 Analysis of proteinase chromogenic substrates

Conduct a comparison of the SCCE, bovine chymotrypsin and cathepsin G of human rights in relation to the influence of inhibitors Aprotinin, hemostasia, zinc sulfate and specificity of the substrate.

The original solution of Meo-Suc-Arg-Pro-Tyr-pNA /S-2586/ prepared in distilled water, a solution of Suc-Ala-Ala-Pro-Phe-pNA /Bochringer, Mannheim, Germany/ in 1-methyl-2-pyrrolidone /the final concentration of solvent in the incubation mixtures 4%/ and hemostatis in dimethyl sulfoxide /the final concentration of solvent in the incubation mixtures 1%/. Cathepsin G of purulent sputum person receive from E. Lotti, Geneva, Switzerland. Source SCCE is KCl-extract dissociated corneocytes corneal layer of the heel surface, obtained as described wounds which are conducted at 37280C in microtiter plates. Total incubation is 135 mm. Each incubation mixture contains Tris-HCl pH 8.0 /final concentration of 0.08 M/, KCl /concentration of 0.2 M/, 100 µl of substrate solution, 25 μl of enzyme source /appropriately diluted in 0.1 M Tris-HCl pH 8.0, and 1.0 M KCl/ and 10 μl of inhibitor solution.

In Fig. 9 A-C, Meo-Suc-Arg-Pro-Typ-pNA /S-2586, initial concentration of 1.2 mm was used as the substrate. In D. initial concentration of both substrates is 1.2 mm.

By the end of the incubation /1.5 hours/ add 125 ál of 10% acetic acid in each cell, and the absorption read at 405 nm, using as a comparison of the incubation mixture without added enzymes. The amount of added enzymes set such that the change of absorption at 405 nm by the end of the incubations would be 0,3 - 0,7.

The results are summarized in Fig. 9, A - D. in order to study the effect of inhibitors, as a substrate using S-2586. The effectiveness of Aprotinin as an inhibitor SCCE and chymotrypsin were high and about the same for these two enzymes. On the other hand, the impact on cathepsin G much less /of Fig. 9 A/. Hemostatic leads to inhibition of all three as the e for SCCE, than for chymotrypsin and cathepsin G /Fig. 9 B/. Zinc sulfate is an effective inhibitor of SCCE, but not chymotrypsin and cathepsin G /Fig. 9 C/. The activity of three enzymes against substrate Meo-Suc-Arg-Pro-Tyr-pNA /S-2586/ and Suc-Ala-Ala-Pro-Phe-pNA compare /see Fig. 9 D/. Because the purpose was to determine similarities or differences between the investigated enzymes, these experiments were performed with only one source concentration for each substrate. While Suc-Ala-Ala-Pro-Phe-pNA are, apparently, a much better substrate than S-2586 for chymotrypsin and cathepsin G, for SCCE opposite is true.

Example 4

Purification of N-terminal amino acid sequence defining chymotrypsinlike enzyme corneal layer

4.1. The selection of SCCE KCl-extracts of corneocytes using affinity chromatography on resolubilization the inhibitor soybean trypsin /SBTI/.

In Fig. 10 shows the results of affinity chromatography on SBTI. Affinity gel is prepared by linking 50 mg SBTI /Bochringer, Mannheim, Germany/ c 12 ml of precipitated Affigel 15 /Bio-Rad, Richmand, CA/, in accordance with the manufacturer's recommendations. The remaining active groups on the gel were blocked by ethanolamine. United KCl-extracts from 100 g /dry weight/ corneal layer of patch is oresti 42 ml/HR continuous recording of the absorption of the eluate at 280 nm. The column was washed with 0.1 M Tris-HCl pH 8, 1 M KCl, up until the eluate absorbance becomes not less than 0.01, and then 10 ml of 0.1 M Tris-HCl pH 8. Stepwise elution of the bound material is carried out with HCl at 1, 10 and 100 mm. Eluent replace, when the absorption of the eluate is reduced below 0.01. Fractions of 3 ml is collected in a test ampoules that contain Tris-HCl pH 8, the total volume of 0.4 ml in quantity calculated so that it was enough to establish the pH of the eluate is above 7. the pH of each fraction immediately check and, if necessary, set about 7 due to the small volumes of 1 M Tris-HCl, pH 8. Analyses of peptide hiraizumi activity with S-2586 /substrate for SCCE/ and S-2288 /substrate for trypsin-like enzymes/ spend by way of example 2.1. The initial concentration of both substrates in the analytical mixtures is 1.1 mm. About 90% S-2586 hydrolysing activity associated with the gel. After stepwise elution of the washed gel with 10 - 100 mm HCl, about 60% of the full S-2586 hydrolysing activity in applied KCl-extract can be distinguished. From the full S-2288 hydrolysing activity of about 20% is related to the affinity gel and 10% can be identified in the eluate.

Fig. 11 shows the analysis of the eluate from SBTI-affinity chromatography with electrophorese samples. Cm. also Egelrud and Lundstrom, 1991 and example 2, 2.2 for details of the experiment. Before preparation for electrophoresis samples are concentrated about 20 times in a centrifuge filtering Ultrafrec MC-filters /sections 10 KD: Millipore, Bedford, MA/ and diluted 10-fold in B.

Fig. 12 is a comparison by SDS-PAGE unrestored and restored samples from SBTI-affinity chromatography.

As follows from Fig. 10 and 11, SBTI-affinity chromatography gives the protein, which has a purity of more than 90% of /as judged by Coomassie-blue-stained SDS-PAGE gels/ with apparent molecular weight of approximately 25 KD in unrestored form, and about 28 KD in restored form. In addition, there is a small Coomassie-blue-positive component with an apparent molecular weight of about 1 KD more than the main component. If demografie gels there is one major and one minor band with the same electrophoretic mobility as the two bands detected on Coomassie-blue-stained gels with unrestored examples. Both of these caseinolytic components can inhibit homestation. In addition, zymography shows small components with facile molecular weights of about 30 P CLASS="ptx2">

4.2. Analysis of N-terminal amino acid sequence SCCE

200 ál fractions of the chromatogram with SBTI-Affigel 15, A1nm 0,2, prepared from SDS-PAGE with the restoration or without him, and treated with 12.5% polyacrylamide gel /thickness 1 mm, slit width 73 mm/. After electrophoresis the selected proteins electrophoretic transfer to Immobilon filter /Millepore/ and dyed with Coomassie blue according to the method Matsuidaira, 1987. A major protein band of cut and processed in an Applied Biosystems 477A pulsed liquid-phase analyzer amino acid sequence connected with 120A PTH analyzer /Applied Biosystems Inc., Foster Gity, CA, USA. Sequencing is carried out in accordance with cyclic programs and chemicals from manufacturers. Initial and re-outputs, calculated with respect to the standard proteins are 25 and 97%, respectively.

The outputs of amino acid derivatives correspond to only one sequenced peptide. For unrecovered samples the outputs were good at stages 1 - 6, but fell to 0 at the stages 7 and 9. The outputs at the subsequent stages were significantly reduced. Although the recovered samples at stages 7 and 9 were not detected derivatives of amino acids, but for the subsequent stages, the DG is put, in positions 7 and 9 are cysteine. However, it was not possible to detect karboksimetilirovaniya cysteine at stages 7 and 9 after recovery and processing Vodokanal acid /100 mm/. The obtained sequence /Fig. 13, a sequence of 1D N 3/ was identical to the restored and unrestored samples.

Example 5

5.1. Getting SCCE-specific monoclonal antibodies

Mice BALB/c /Bomholtgaard, Denmark/ injected approximately 30 μg of native SCCE, purified by the method of example 4.1, in complete Freund's adjuvant /Difco Laboratories, Detroit, MI/ by subcutaneous injection. Injection with the same amount of SCCE in incomplete Freund's adjuvant /Difco Laboratories, Detroit, MI/ repeat in a month. 4 months after the first injection, one mouse intravenous booster injection in the next 3 days, 30 μg of antigen per injection. Hybridoma get in the way described by Carlsson et al., 1985, with cells of the SP2/0 myeloma cell line /ATCC CRL 1581/. Identification of antibodies that react with purified SCCE drugs, are using ELISA. The supernatant culture fluid of positive clones analyzed further using Western blot turns after SDS-PAGE. Clones that produce antibodies that react with SCCE in this test, multiply the mouse is soba, described in Carlsson et al., 1985. Get two suitable antibodies, oab Te4b and oab TE9b, and both are classified as LgGo-Kappa.

Characterization oab Te4b and oab TE9b using immunoassay and Western blot turns is shown in Fig. 14.

Fig. 14a /strip 2/ is Coomassie blue stained SDS-PAGE gel with concentrated KCl-extract dissociated corneocytes heel surface obtained by the method of example 3.1. Sample cialiswhat within 4 hours against 0.1 M sodium acetate, pH 4, and concentrated approximately 100-fold by ultrafiltration before electrophoresis. Fig. 14a /band 3/ is getting SCCE, purified by the method of example 4.1.

Fig. 4b presents the results of the experiment immunosurgery, in which antibodies were incubated with KCl-extract corneocytes, and then allocated through insolubilizing protein A. Re solubilization and dissociatively complexes of antibody-antibody were analyzed using demografie according to the method of example 2.

250 μl of KCl-extract dissociated corneocytes heel surface, which were 5-fold concentrated by ultrafiltration, dialist against superyoung phosphinothricin 10 mg/ml, mixed with 10 μl of a solution of antibody or superyoung phosphate saline, and incubated for 15 hours at 4oC. 25 μl of precipitated protein Sepharose /Pharmacia, Uppsala, Sweden/ add then in ampoules, and continue incubation with gentle shaking at room temperature for 2 hours. Gel allocate by centrifugation and washed five times with 1 ml of 0.05% Tween 20 /Sigma, St. Luis, MO/ in 0.05 M Tris-HCl, pH 7.5, 0.5 M NaCl. After the final washing, the gel is extracted with 100 μl of sample buffer Laemmli without a reducing agent for 1 hour at room temperature. These extracts are purified by centrifugation and applied to the gel.

oab TE4b and TE9b, both precipitated caseinolytic enzyme, with the same Mg, and purified SCCE, and the corresponding main caseinolytic enzyme in KCl-extract. Antibodies precipitated smaller caseinolytic enzymes in the extract from Mg to about 30 kDa, which has been shown to be inhibited by leupeptin, inhibitor of trypsin-like serine proteases. In addition to the 25 kDa caseinolytic enzyme antibodies, apparently, bind small proteinopathies component with Mg of about 80 kDa. This component is usually present in KCl-extracts of corneocytes heel is x fresh tissue /T. Egelrud, unpublished observations/.

He is not present in SCCE-preparations isolated using affinity chromatography, and may be present in the products of aggregation. Failed to locate a matching component that reacts with antibodies to immunoblot.

On immunoblot SDS-PAGE gels, conducted in non conditions /Fig. 14c/ oab TE4b and TE9b react with a component present in KCl-extracts of corneocytes heel surface /Fig. 14 with lanes 2 and 4/ and treated in SCCE-drugs /Fig. 14c, lanes 3 and 5/, and they both have the same Mg as the main selected protein and the main caseinolytic component on zymogram. These antibodies were directionspanel samples, which were restored in the presence of SDS, which implies that they are directed against conformation-dependent epitopes.

In addition to the basic protein with Mg of about 25 KD in unrestored form, purified SCCE-the product contains a small Coomasie blue component with Mg of about 26 KD /unrestored; see example 3/. On zymogram corresponding caseinolytic component is present and can be ingibirovany homestation the same way for the main 25 KD caseinolytic is about to see this small component, reactive with antibodies to immunoblot. Similar results /not submitted/ received with polyclonal rabbit antibodies generated against Coomassie blue-positive components, selected by preparative electrophoresis. The exact ratio between the two proteins with SCCE-like activity and apparent immunological cross-reaction is unknown.

5.2. Antibodies specific polyclonal SCCE.

5.2.1. Chicken anti-SCCE

45 mcg SCCE selected with SBTI-affinity chromatography according to the method of example 4.1, 0.2 ml of 0.1 M Tris-HCl thermodenaturation for 60 minutes at 60 ° C and homogenized in an equal volume of adjuvant-blockers /Difco Laboratories/.

The resulting emulsion is injected subcutaneously in Derco chickens, approximately 20 weeks of age, which was selected samples of blood for the preparation of pre-immune serum. Chickens have introduced additional subcutaneous injections of the emulsion obtained as described previously, but with incomplete adjuvant's adjuvant and with 30 μg of purified, thermodenaturation SCCE /full amount of each emulsion 250 ál/ after 3, 5 and 7 weeks. Chicks collected blood 2 weeks after the last injection. This blood is immediately mixed with 2 volumes of a solution Anyplace anti-SCCE, selected for further research, use 1/2000 dilutions in experiments with Western blot turns. Cm. Fig. 17, the example 8 to illustrate the specificity of the antisera.

5.2.2. Rabbit anti-SCCE

SCCE selected with SBTI-affinity chromatography, subjected to SDS-PAGE without recovery, as described in example 4.1, on the gels with a thickness of 15 mm in accordance with the data Laemmli, 1970. The main protein band, which was previously shown to be an SCCE was visualized by using a method of dyeing a chloride of copper, in accordance with the work of Lee et. al., 1987, and it cut out. After removal of chloride of copper with EDTA according to the method of Lee et. al. the gel slices homogenized bateriafina phosphate saline solution. Samples of homogenised pieces of gel are suspended in equal volume of adjuvant's adjuvant. Approximately 30 μg of pure SCCE, thus obtained in complete Freund, injected subcutaneously rabbit. After 3, 5 and 7 weeks for repeat injection with the same amount of SCCE, but with incomplete adjuvant. The blood of a rabbit taken two weeks after the last injection.

Received rabbit anti-SCCE /D-5/ use in the dilution of 1/500 - 1/1000 in experiments with immunoblots with alkaline phosphatase, conjug of Western blot turns linked second antibody is determined by the way Blake et. al., 1984 (applies to examples 5, 8 and 9).

Rabbit anti-SCCE Bo-1 are the same way, but with SCCE, which were restored before SDS-PAGE, as antigen.

5.3. Immunohistochemical study with monoclonal antibodies

In immunohistochemical studies with SCCE-specific monoclonal antibodies, SCCE can be detected in high suprabasal cells keratinizing squamous epithelium of the epidermis, the inner shell of the roots of the hair follicle, solid plate/, but not in nitratenitrogen squamous epithelium /internal root sheath of the hair follicle, mucus lips and inner cheeks/. SCCE can specifically Express in keratinizing squamous epithelium. In addition, it was found that the SCCE is expressed in high supersatellite cells of human epidermis reconstructed in vitro and grown at the interface air-water. If the environment add retinology acid concentration, which stimulates the proliferation of keratinocytes, but inhibits the formation of the corneal layer, SCCE is no longer expressed. This suggests that SCCE expression may be part of a program of differentiation of the epidermis.

Example 6

Cloning and sequencing of cDNA encoding human SCCE

Restriction enzymes get from Promega, Madison, MI, and TAQ polymerase from Perkui-Elmer-Cefus, Norwalk, CT. The cDNA library keratinocytes gt II man

derived from mRNA derived from keratinocytes of the epidermal origin of the adult, obtained from Clontech Laboratories, Polo Alto, CA (N directory HL 1045 b). Initially, the library sceneroot anti-SCCE rabbit polyclonal serum D-5 and Bo-1 (see example 5.2.2.). As Bo-1 polyclonal anti-SCCE serum gives high background signals, it is excluded from interesting skanirovaniya at an early stage. Using D-5 anticigarette, the number of immunoreactive plaques were enriched as proposed for true-positive plaques. No reactivity was not observed with monoclonal antibodies oab 4 and oab 9 for none of the plaques. Intensive characterwidth between different plaques. The presence of such partial similarity indicates that platelets contain homologous DNA inserts from the same cDNA sequence. As it is not possible to define the "fingerprint" of possible SCCE cDNA sequence, a strategy had to be modified.

Plaques were skanirovaniya in E. coli y 1090 (Clontech) by plaque hybridization using as a probe degenerate synthetic oligonucleotide. The oligonucleotide probe was designed on the basis of experimentally defined aminoterminal sequence of native SCCE enzyme as described in example 4.2. The most reliable part of the amino acid sequence Ile-Ile-Asp-Gly-Ala-Pro (sequence ID No. 3 aa 1 - aa 6) was selected to construct a synthetic 17-dimensional oligonucleotide probe 5'-ATHATHGAYGGNGCNCC-3' /H=A or C or T; Y=C or T; N=A or C or G or T/ marked SYM3067, sequence ID No. 4. Oligonucleotide probe synthesized using Ismap 200A DNA synthesizer using phosphoramidite method in accordance with the instructions.

E. coli Y 1090 bacteria grown overnight in LB in the environment /Sambrook et al. , 1989/, containing 0.2% maltose and 10 mm MgSO4, 0.4 ml of the culture is mixed and then diluted with Biblio 6 ml soft agarose /a 0.75% agarose in LB and 10 mm MgSO4/. Soft agarose mixture was poured on 10 A 150 mm wafer. Plates are incubated for 5 hours and placed overnight at 4oC. In General, all plates contain approximately 4105plaques.

For immobilization of plaques per plate cover NEN Du Pont Colony /Plague Screen membranes /Du Pont. Wilmington, DE/ for two minutes. The obtained membrane is dipped twice for 2 minutes in 0.5 M NaOH, twice for 2 minutes in Tris-HCl pH 7.5 and allowed to dry. Then these membranes are used for hybridization experiments, as will be indicated hereinafter. These membranes are pre-hybridized in 10% dextran sulfate, 1M NaCl, 1% SDS solution containing 100 mg/ml denatured sperm DNA herring /Sigma, St. Louis MO/, for 5 hours at 65oC. as a probe used SYM 3067, [-32P] dATP-labeled using T4 polynucleotide kinase /Promega, Madison, W1/ and add it to prehybridization mixture. Hybridization of conduct within 12 - 18 hours at 42oC.

After hybridization the membrane was washed four times for 5 minutes in 2xSSC at room temperature, twice for 30 minutes in 2xSSC, 1% SDS at 42oC, and finally in 0.1 SSC at room temperature for 30 minutes. These membranes autoradiography on x-ray film /Hyperfilm-MP, Amersham, UK/. In perfect same probe and method as described previously. After the procedure, re-screening identify two positive plaques. Two selected plaques once again clear, and the sizes of the inserts determined through PCR, using as primers SYM 1600 and SYM 1601, and selected plaques as a matrix. These two primers complementary left and right shoulders gt 11 phage, respectively. Amplificatory DNA fragment of about 0.9 kb, derived from A phage 6.2.2, digest then EcoR1 and clone into EcoR1-digested pUC19 /Pharmacia, Uppsala, Sweden/, pS 496. This cloned fragment is then subjected to partial sequence analysis using primers sequences complementary pU C19. The nucleotide sequence determined using T7 Sequeira sets /Pharmacia, Uppsala, Sweden or USB, Cleveland, Ohio/.

Broadcasting the obtained DNA sequence leads to an amino acid sequence that is homologous experimentally particular protein sequence.

However, this sequence does not contain a translational start codon. For isolation of full length cDNA, the DNA fragment can be distinguished in an agarose gel and used as a probe, providing hybridization in hard condition which we add in the amount of 3 ml per gram of the gel and placed in a bath of boiling water for 7 minutes, to melt the gel and denaturing DNA. Then the vial is transferred to a water bath at 37oC for at least 10 minutes. In the reaction mixture for introduction of labels add volume DNA/agarose solution containing approximately 25 ng DNA. /This is in accordance with the instructions of the supplier/.

To obtain full length cDNA, a cDNA library again sceneroot twice, using the same methods as described previously, except that the hybridization lead to severe conditions at 65oC. In these experiments identify and allocate 45 individual positive plaques that first sceneroot using PCR, using SYM 1600 /5'-GTG GCG ACG ACT CCT GGA-GCC-3'; sequence ID No. 5/, or SYM 1601 /5'-ACA CCA GAC CAA CTG GTA ATG-3' /sequence ID No. 6/ combined with SYM 3208 as PCR primers for the identification of plaques containing the complete 5'- open reading frame. SYM 3208, 5'-TGGGTGGGAGCCTCTTGCACA-3', sequence ID No. 7 at least partially complementary to the 5' part of the SCCE cDNA was constructed on the basis of Information about the DNA sequence obtained from pS 496. After this screening, were selected 4 phage for further analysis. For sequence analysis, the obtained PCR amplificatoare fraud, one of these phages, 205.2.1 contains the full length of the insert.

DNA from phage isolate 205.2.1 get in the way Sambrook et al., 1989, and DNA preparation is digested by EcoR1. Digested DNA allocate electrophoresis on agarose gel, and a fragment of about 1 kb isolate and clone into EcoR1-digested pUC19. The obtained plasmid denote pS 500 /Fig. 15/. The complete nucleotide sequence of the cDNA fragment determined as described previously. As primers for sequencing reactions using specific oligonucleotides, complementary pUC19 or SCCE sequence. The nucleotide sequence /sequence ID No. 1/ contains an open reading frame sufficient to encode the complete amino acid sequence of the SCCE protein precursor, which consists of 253 amino acids, including the signal peptide and propolypeptide /sequence ID No. 2/.

As was discovered, another phage, named 106.1.2, contains SCCE cDNA sequence that lacks the 5'-noncoding sequence, and the first three codon. This insert has identified as the 954 bp EcoR1 fragment and clone in EcoR1 linearized pUC19, resulting in plasmid pS 498. This plasmid is sequenced partially.

Tr who was 5'-noncoding sequence and seven nucleotides broadcast area. This cDNA insert has a longer option 3'-noncoding area stretching from 1057 bp in the opposite direction from the stop codon. This 1884 bp EcoR1 fragment isolated and clone in EcoR1 linearized pUC19. The obtained plasmid is sequenced completely and denote pS 501.

Example 7

Definition SCCE mRNA in human epidermis

Obtaining total RNA from human epidermis

This is carried out in accordance with the methodology Chomcgynski and Sacchi, 1987. Abdominal skin of a healthy person get with plastic surgery. Immediately after you remove it cooled on ice. Within less than 15 minutes epidermis secrete, scraping with a scalpel dipped in a solution of D /Chomcgynski and Sacchi, 1987/ and homogenized in a glass homogenizer. Then do everything according to the scheme Chomcgynski. The precipitated total RNA stored at -20oC in 70% ethanol until further analysis.

Obtaining information RNA

500 µg total RNA of the epidermis is treated using a set of Poly A Tract /Promega/ in accordance with the instructions of the suppliers.

RNA electrophoresis and blotting

Agarose gels /1.4 percent/ cook with 0.66 M formaldehyde in 1MOPS buffer and 0.6 g/ml ethidiumbromid /Sigma, St. Loo MO/. the mRNA corresponding to 100 µg total RNA, restwood before applying. RNA markers /BRL, Geithersburg, MD/ treated similarly. After electrophoresis the gels immersed in distilled water for 5 minutes, and then in 50 mm NaOH for 30 minutes in 0.1 M Tris-HCl pH 7.5 for 30 minutes. Carry blotting membrane Gene - Screen Plus /NEN Du Pont, Wilmington, DE/ s Vacu-Gene equipment /Pharmacia, Uppsala, Sweden/ 1 hour in 10SSC. Then the membrane is washed 3SCC, dried over night and heated for 2 hours at 80oC. RNA present on the membranes in UV-light.

cDNA probes

Plasmid pS501, obtained as described in example 6, digest Hinc II and Bgl II. This cDNA contains one Hincll site at bp N 1060 and one Bgl 11 website at bp N 1715. 1070 bp fragment /Hinc II site in the pUC19 multiple cloning site of the endogenous Hinc II site/ includes SCCE encoding section in addition to the 7 bp from the 5' end and noncoding site, including the site of polyadenylation at bp 944-951, which is common to all SCCE-cDNA, which were highlighted. Fragment 655 bp Hinc II-Bgl II, which does not contain a poly-A-tail is unique for SCCE cDNA 108-1-2. These fragments were separated by electrophoresis on agarose and were used to obtain the32PdCP-labeled probes using the Multiprime kit for the introduction of labels in DNA /Amersham, Buckinghamshire, UK/.

Hybridization

Membrane cipater herring 0.1 mg/ml for 3 hours. Hybridization lead to the same solution at 60oC during the night. Leaching is carried out h minutes at 60oC in 1% SDS in 2SCC and within 3 hours in 0.1 SCC at room temperature. Then the membrane is subjected to autoradiography.

The results:

It is possible to demonstrate the presence in the human epidermis two mRNA species with sizes of about 1.2 kb and 2.0 kb, respectively, /Fig. 16/. This is in good accordance with the evidence obtained in experiments on cloning, where they found two types of cDNA.

Example 8

Expression of recombinant SCCE in E. coli

Construction of the plasmid pGEX-2T/SCCE

1. Semantic PCR primers

1.a.

CGTGGATCCATCGAAGGTCGTATTATTGATGGCGCCCCATGT /SYM 3367, ser. ID No. 8, highlighted the 3'part of the encoding II-terminal amino acids IIDGAPC active native SCCE, 5'-portion with Bam H1 site, and an additional sequence encoding a site of factor Xa, IEGR.

1. b. CGTGGATCCATCGAAGGTCGTTTGGAAACTGCAGGAGAAGAA /SYM 3368, sequence ID No. 9, highlighted by a 3'-portion, corresponding to base pairs 76-96 in full SCCE-cDNA sequence that encodes the amino acid sequence LETAGEE, 5'-portion as in 1a.

2. Antisense PCR primers TGATCCTCTGAGCTCTCCTG /SYM 3371; complementary pairs based outermost pS 498 /example 6/ PCR-amplified with primers 1a/2 1b/2. These products are purified by extraction with phenol and precipitated with ethanol, digested by Bam H1/ Sac1 and purified by electrophoresis in agarose. Then clone in TG2 cells in pGEX-2T /Pharmacia/, digested by Bam H1/EcoR1 with 3' 673 base pairs SCCE 106-1-2, obtained by digestion of pS 498 Sac1 and EcoR1. From bacterial clones that were used to study the expression plasmids /pS 510 encoding a native II terminal, next to the site of factor Xa, and pS 511, the coding propeptide following the site of factor Xa/, were isolated, and the nucleotide sequence corresponding to the inserts obtained from PCR products were checked by way of dideoxy-chain termination using a set of T7 for sequencing /Pharmacia, Uppasala, Sweden/.

Studies of expression

Culture (ongoing throughout the night)

TG cells with pS 510 and pS 511 in LB medium containing 50 μg/ml Carbenicillin /Sigma St. Louis MO/, diluted 10-fold in fresh medium and grown for 3 hours at 37oC. IPTG /Sigma, St. Luis, MO/ add to final concentration of 0.1 M, and the culture is grown at 37oC for another 3 hours. Bacterial precipitation is treated with ultrasound in PBS 1% Triton X-100 /Sigma, St. Louis, MO/. After centrifugation at 10,000 x g for 15 minutes, the supernatant of the anti-serum.

Large amounts of IPTG-induced protein with about 50 Mg CD /pS 510/ and 52 KD /pS 511/ SCCE-like immunoreactivity were detected in the sediments, not soluble in PBS-Triton X-100 (see Fig. 17/.

The supernatant after ultrasonic treatment in PBS-Triton X-100 containing GST/SCCE fusion proteins of the same size as in the insoluble sediments, but their number is small compared with the number in the insoluble precipitation.

These results show that it is possible to Express the SCCE as a fusion protein with GST, and the sequence corresponding to the specific protease sites of cleavage in pre-Pro - SCCE amino acid sequence will make it possible to repeat this experiment in order to obtain recombinant SCCE in bacteria. The resulting protein can be solubilisate in the urea or guanidinopropionic, and then cleaned using ion-exchange chromatography due to the high isoelectric point of the SCCE. The purified protein can be denaturiruet, dialigue against buffers with a low concentration of denaturing agent, and then split factor Xa to release GST polypeptide of the SCCE or Pro-SCCE. The fusion proteins GST/SCCE can also be used as immunogens to get SCCE-Spa is combinatori SCCE person in mammalian cells

To create the expression vector to obtain a recombinant SCCE human cDNA consistently isolated from the plasmid pS500 in the form of 897 bp EcoR1/Dral fragment. This fragment subcloning in EcoR1 and Sma1-digested pUC19, getting pS502. Then plasmid pS502 digested by EcoR1 and Sal 1 to highlight the cDNA sequences of SCCE in the form of a 0.9 kb fragment, which again subcloning in pUC19 option that does not Hind111 site, receiving plasmid pS503. This pUC19 option receive the digestion of pUC19 with Hind111, filling, using an enzyme maple and again ligera. To facilitate cloning into the expression vector, Hind111 site is introduced into the 5'-end cDNA SCCE. Do it, digesting pS503 EciR1 and embedding the linker, which turns the Hind111 site, SYM3603

5'-AATTGTGGAAGCTTCCAC-3', sequence ID No. 10. The obtained plasmid that carries the coding for the protein part of the SCCE cDNA with Hind111 site at the 5' end and Sal 1 site at the 3' end, respectively, denote pS505.

The final expression vector get ligera three different DNA fragment. First, pS505 digest Hind 111 and Sal 1, and produce a fragment of 0.9 kb.

Then, to obtain the distal regulatory element located in the opposite direction, mouse metallothionein, follow the processing mRNA, and plasmid sequences, pML2d, to ensure the selection and replication in E. coli /Waldenstrom et al., 1992/ vector pS147 digest SaC1 and Sal 1, and produce a fragment of about 12 kb.

Thirdly, to highlight the proximal promoter of the mouse metallothionein, plasmid pS42, in which native Bgl 11 is located in the leader sequence, was turned into a Hind 111 website, digest Sac 1 and Hind 111, and produce a fragment of approximately 220 bp.

Ligation of these three elements results in the expression vector SCCE pS507 /see Fig. 18/.

The expression vector pS507 together transferout with the vector containing the gene of resistance to neomycin, managed 5' long terminal repeat sarcoma virus Harley, and signals from SV40 polyadenylation /Lusky and Botchan, 1984/, murine C127 cells /ATCC CRL 1616/. Experiments transfection is carried out in accordance with the method of calcium-phosphate deposition /Graham and Van der bb, 1973/.

Cells cultivated in Ham's F12 /Dulbecco''s modified Eagle medium /DMEM; Gibco, BRL, Gitersburg, MD/ /1:1/, supplemented with 10% serum, fetal calf /HyClone, Logan, UT/. Clones of cells resistant to neomycin, selected with 1.5 mg/ml G418 /Gibco-BRL/ and after 10 to 15 days after the selection of resistant cell clones identify and vydeleny SCCE gene prepare total RNA from selected cell lines. Total RNA derived from C127 cells and secrete on 1% formaldehyde-agarose gel, transferred to a nitrocellulose membrane and hybridized with SCCE probe, labeled32P. This probe represents 1070 bp Hinc 11 fragment SCCE cDNA allocated Hinc 11 digesting pS500, and agarose electrophoresis. The experimental procedure is consistent with the instructions in Ausubel et al., 1992. Experiments on Northernblues and hybridization with32P-labeled SCCE cDNA show that recombinant SCCE mRNA can be detected in several cell lines containing SCCE vector, pS507. Hybridization was not detected in control samples derived from C127 cell line containing an identical vector, except SCCE cDNA /Fig. 19/. Size 1.4 kb corresponds to the expected size.

Samples of conditioned cell culture medium is collected and analyzed in Western blot turns. SDS-PAGE carried out according to the method Lemli /1970/ and for Western blot turns as detecting antibodies using chicken anti-native SCCE. Labeled alkaline phosphatase anti-chicken IgG /Sigma, St. Louis, MO/ used for enzyme labels. The results obtained are shown in Fig. 20.

To analyze exprate control samples have total RNA from both retrospectively C127 cells, and C127 cells, transfection expression vector pS147. Vector pS147 similar vector pS507 except that it contains the cDNA for bile-salt-stimulated lipase person /Nilsson et al., 1990/ instead of SCCE cDNA person. RNA receive according to the method of Ausubel et al., /1992/. Experiments Northernblues and hybridization32P-labeled cDNA SCCE show that recombinant SCCE mRNA of approximately 1.4 kb is detected in C127 cells containing SCCE vector, pS507 /Fig. 19/. Hybridization was not detected in control samples derived from C127 cell lines containing pS147 or retrospektywnie C127 cells. Length recombinant SCCE mRNA corresponds to the expected value.

Samples of conditioned cell culture medium is collected and analyzed through SDS-PAGE and Western blot turns. The blots show the way Blake et al., 1984. The results obtained (see Fig. 20/ show that C127 cells containing pS507 produce three proteins that demonstrate the reaction of all available polyclonal rabbit and chicken SCCE antibodies and with anti - SCCE monoclonal obtained by the method of example 5. Recombinant SCCE reactive proteins demonstrate the apparent molecular weight of about 1 kDa more, jectively. When comparing the proposed SCCE amino acid sequences with experimentally obtained NH-2 by the end of native SCCE person and with sequences of other chymotrypsin-like proteases, we can conclude that recombinant SCCE obtained in C127 cells, is its Pro-enzyme form. Sequence data show that this Pro-enzyme can be activated by proteolytic cleavage by C-Terminus of lysine in the sequence . . .AQGDKIIDGAP..., where the underlined sequence is the NH-2 sequence active native SCCE person /sequence ID N2, aa 5 aa 6/.

Example 10

Isolation and characterization of recombinant SCCE

Selection

1.5 mg monoclonal antibodies TE4b against native SCCE, combined with 1.5 ml Spug-activated sepharose /Pharmacia-LKB Biotech., Uppsala, Sweden/, using the method recommended by the manufacturer. 40 ml of medium containing SCCE, filtered through a 0.45 μm filter and then injected into the column. The column is washed several times with 10 mm phosphate, 150 mm NaCl, pH 7.2 and then elute with 0.1 M glycine-HCl, pH to 2.5. Suirvey protein is immediately neutralized by adding a 0.1 volume of 1 M Tris-HCl, pH 8.0.

Activation

Recombinant SCCE /4.6 mcg the Cena /mass ratio of 10:1/ at 37oC. Samples /50 μl are taken after 20 minutes, 1 hour, 3 hours and 20 hours, and 5 μl of 10 μm /4-lidinopril/methanesulfonyl /APMSF; Boehringer Mannheim, Germany/ add to the end of the reaction.

Activity split SCCE analyzed by non SDS-PAGE on casein gels according to the method of example 2.2 Identity derived derived forms SCCE was analyzed by reducing SDS-PAGE with subsequent Western blot turns, using chicken anti-native SCCE with the subsequent introduction of a label alkaline phosphatase anti-chicken IgG and nitrobluetetrazolium and 5-bromo-4-chloro-3-indolylacetic as a substrate for alkaline phosphatase.

N-terminal sequencing

Afinno cleaned /as indicated/ SCCE, about 35 μg, was subjected to slot blot using install Bio-Dot SF. /Bio Rad, Richmond, CA/ on the membrane Immobilon /Millipore, Bedford, MA/. Then the membrane is washed several times with distilled water to remove all Tris and glycine. Part of the membrane, which was associated protein, cut out and is sequenced using Applied Biosystems /Foster City, CA, 477A pulsed liquid-phase sequencing machine connected with 120A PTH analyzer. Sequencing is carried out, using the cyclic prog glu-glu-ala-gln-gly-asp, which corresponds to amino acids -7 -2 sequence ID No. 2. As can be concluded from this result, the signal peptide consists of 22 amino acids and is based on the N-terminal amino acid sequence of the native active SCCE and propeptide consists of seven amino acids.

Deglycosylation

Purified recombinant SCCE /5 ág/ and native SCCE /20 mcg/ boiled for 3 minutes in 20 μl of 0.5% SDS and 0.1 M mercaptoethanol. Samples diluted nutrifaster buffer, pH 8.6 and Nonidet P-40 to a final concentration of 0.2 M and 1.25%, respectively. Add N-glycosidase F/Boehringer Mannheim/ for recombinant protein of 0.6 units, and for the native protein 1.2 units of the enzyme, and the reaction mixture is incubated over night at 37oC. the Final concentration of SDS in the analyzed sample was 0.17%. Processed N-glycosidase FSCCE analyze 8 - 18% SDS-PAGE with subsequent Western blot turns, as indicated previously. The results obtained demonstrate the recovery of the apparent molecular weight of the two upper bands, whereas the apparent molecular weight of the lower band does not change /Fig. 23/. This shows that recombinant SCCE produced in C127 cells, exists in two N-gli is camping for active native SCCE /Fig. 23/.

Example 11

Compositions containing SCCE

Such compositions can be obtained in accordance with conventional pharmaceutical techniques, including mixing the active compounds with other ingredients. All percentages are given by weight.

Compositions which contain more than one active connection, also included in the scope of the invention. The following examples should be considered as also including more than one active connection. Similarly, the term "SCCE" can be replaced by "Pro-SCCE". Thus, the scope of the present invention includes compositions which contain as SCCE and Pro - SCCE.

SCCE = native or recombinant chymotrypsinlike enzyme corneal layer, optionally in combination with other active compounds

q.S. = guantum satis (as needed)

Cream the butter/water, %:

SCCE IS 0.01 - 20

Polysorbate 80 and 0.5

Emulsifying wax - 5

Mineral oil - 4

Dimethicone - 1

Literallayout - 6

Antioxidant - q.S.

EDTA - 0,1

Preservative - q.S.

Glycerol 85% - 4

Propylene glycol - 7

The agent regulating the pH of 0.01 - 10

Water - 65 - 76

Examples of modifiable factors, Antioxidants, chelating agents, whom the agent and the other related to the excipients/.

Cream water/oil, %:

SCCE IS 0.01 - 20

Cetyl alcohol - 0,5

Lanolin - 5

White petrolatum - 10

Mineral oil - 45

Antioxidant - q.S.

EDTA - 1

The agent regulating the pH of 0.01 - 10

Preservative - q.S.

Water - 15 - 25

Examples of modifiable factors, antioxidants, chelating agents, preservatives, emulsifying systems /the ratio between the oil phase and water phase and the content of the emulsifying agent and the other related to the excipients/

Ointment, %:

SCCE IS 0.01 - 20

Lanolin - 15

Petrolatum - 58 - 68

Mineral oil - 15

Dimethicone - 2

Antioxidant - q.S.

Examples of modifiable factors: antioxidants.

Liniment, %:

SCCE IS 0.01 - 20

Emulsifying wax - 4

Literallayout - 3

Mineral oil - 15

Polysorbate 80 - 0,6

Glycerol 85% - 3

Propylene glycol - 5

Antioxidant - q.S.

EDTA - 0,1

Preservative - q.S.

Water - 59 - 69

Examples of modifiable factors, antioxidants, chelating agents, preservatives, emulsifying systems /the ratio between the oil phase and water phase and the content of emulsifying agents and other related to the excipients/

Gena resin - 5

EDTA - 0,1

Preservative - q.S.

Water - 59 - 74

Examples of modifiable factors: gelling agents, antioxidants, chelating agents, preservatives.

Aqueous solution, %:

SCCE IS 0.01 - 20

The agent regulating the pH of 0.01 - 10

Cetyl alcohol - 4

Propylene glycol - 5

Preservative - q.S.

Antioxidant - q.S.

EDTA - 0,1

Water - 37 - 89

Examples of modifiable factors, antioxidants, chelating agents, preservatives, degreasing agents, moisturizers.

The solution in ethyl alcohol, %:

SCCE IS 0.01 - 20

The agent regulating the pH of 0.01 - 10

Propylene glycol - 5

Cetyl alcohol - 3

Antioxidant - q.S.

EDTA - 0,1

Ethanol - 50 - 95

Examples of modifiable factors, antioxidants, chelating agents, moisturizers.

Suspension, %:

SCCE IS 0.01 - 20

Carbomer - 0,5

Cellulose resin - 0,5

Polysorbate 80 - 0,1

Propylene glycol - 5

Ascorbic acid is 0.05

Cetyl alcohol - 4

Polysorbate - q.S.

EDTA - 0,1

The agent regulating the pH of 0.01 - 10

Water - 72 - 80

Examples of modifiable factors, antioxidants, chelating agents, preservatives, suspendresume agents.

Examples of modifiable factors: antioxidants, base pastes.

Patch, %:

SCCE IS 0.01 - 20

Cutina zm - 70 - 80

Ministerului alcohol - 5

Castor oil - 2

White beeswax - 10

White petrolatum - 3

Antioxidant - q.S.

Examples of modifiable factors: antioxidants, the basis of the patch.

Spray /manual/, %:

SCCE IS 0.01 - 20

The agent regulating the pH of 0.01 - 10

Ethanol - 30

Glycerol 85% - 5

Propylene glycol - 5

Cetyl alcohol - 3

Antioxidant - q.S.

EDTA - 0,1

Preservative - q.S.

Water - 22 - 57

Examples of modifiable factors, antioxidants, chelating agents, preservatives, degreasing agents, moisturizers.

The solution for aerosol spray, %:

SCCE IS 0.01 - 20

The agent regulating the pH of 0.01 - 10

Isopropylmyristate - 3

Propylene glycol - 5

Ethanol - 48 - 92

Propellant - q.S.

Examples of modifiable factors: degreasing agents, moisturizers.

Spray - foam aerosol, %:

SCCE IS 0.1 - 20

Wax - 3

Ethanol - 50 - 55

Antioxidant - q.S.

EDTA - 0,1

Water - 20 - 35

Propellant - q.S.

Examples of mutable fact is - 20

Derivatives of cellulose - 1 - 3

Twin60 - 1,0

Literallayout - 2,5

Potassium sorbate - 0,2

Antioxidant - q.S.

EDTA - 0,1

Preservative - q.S.

The agent regulating the pH of 0.01 - 10

Water - 50 - 95

Propellant - q.S.

Examples of modifiable factors, antioxidants, chelating agents, preservatives, emulsifying systems /the ratio between the oil phase and water phase, and the content of emulsifying agents and other related to that of excipients/.

Shampoo, %:

SCCE IS 0.01 - 20

Nutriceuticals - 40

Cetyl alcohol - 3

Foaming agent or air conditioning - 3

Sodium chloride - 2

Antioxidant - q.S.

EDTA - 0,1

Preservative - q.S.

Water - 43 - 53

Examples of modifiable factors: the Foundation of shampoos, antioxidants, chelating agents, preservatives, moisturizers, conditioners.

The body shampoo, %:

SCCE IS 0.01 - 20

Nutriceuticals - 40

Cetyl alcohol - 4

Foaming agent or air conditioning - 3

Pearlescent agent - 10

Preservative - q.S.

Antioxidant - q.S.

EDTA - 0,1

Water - 40 - 50

Examples of modifiable factors: the Foundation of shampoos, antioxidant - 20

1-hydroxyethane-1,1-Diaspora acid - 0,2

Glycerin - 0,8

Sodium soap of coconut oil and tall oil - 88 - 98

Supplements - 0,7

Examples of modifiable factors: moisturizers, Foundation for soap.

Powder, %:

SCCE IS 0.01 - 20

Talc - 65 - 70

Kaolin - 6

Titanium dioxide - 2

Calcium carbonate - 8

Magnesium stearate - 3

Corn or wheat starch - 5 - 10

Example modifiable factors: base powders, preservatives, mass ratio.

The conditioner for hair, %:

SCCE IS 0.01 - 20

Cetyl alcohol - 2,2

Alkyltrimethylammonium - 1,25

Octyldodecanol - 1

Citric acid - 1

EDTA - 0,1

Preservative - q.S.

Antioxidant - q.S.

Water Up to 100

Examples of modifiable factors: air conditioners, preservatives, chelating agents, antioxidants.

In a similar way it is possible to prepare a composition for a surface coating containing a compound that can inhibit or enhance the activity of SCCE.

Example 12

Activity on digestion of desmosomes recombinant SCCE

The corneocytes, containing intact desmosome, removed from the skin, cutting off strips to deeper the display 1 M sodium chloride at 4oC to solubilize the characteristic proteases, and then intensively washed with incubation buffer /0.1 M Tris-HCl pH 8.0/ removal engenni proteolytic activity of the drugs. Incubation lead in 0.1 M Tr pH 8.0 with 10 μg of recombinant SCCE /or without/ within 24 hours at 37oC. Proof desmosomal digestion due to enzyme is determining levels desmosomes marker protein desmoglein 1 /DG1/. Select from scales, extragere 8 M urea /2% SDS/ -mercaptoethanol buffer with subsequent cleaning of DG-1 glycoprotein using concanavalin A-affinity chromatography. The eluate concanavalin fractionary due to SDS-PAGE and electrophoretic transfer to PDVF membrane for Western blot turns, DG1 identify, using specific anticigarette, and determine, using the increase in chemiluminescence. The results are shown in table 4.

The results presented in table 4 show that recombinant SCCE able to break down the intercellular cohesive structures in the corneal layer analysis in vitro.

Example 13

The effect of inhibitors on the activity of recombinant SCCE

Examine the effect of inhibitors Aprotinin, himo the results presented in table 5.

As presented earlier in table 5, Aprotinin, hemostatis and zinc ions inhibit S-2586 gidrolizuemye activity of recombinant SCCE in the same way as the native enzyme.

1. The selected nucleic acid, characterized in that it contains a nucleotide sequence represented by sequence ID No. 1, or its complementary strand.

2. A plasmid, designated pS 500 DSM 8281, characterized in that it has a nucleic acid on p. 1 encoding a precursor polypeptide that has activity chymotrypsinlike enzyme corneal layer (SCCE).

3. A replicable expression vector, denoted by pS 507 DSM 8282, characterized in that it has a nucleic acid on p. 1 encoding a precursor polypeptide having SCCE activity.

4. Line of mouse cells With 127 transformed replicable expression vector under item 3, the producer of precursor polypeptide with the activity of SCCE.

5. Substantially pure polypeptide characterized in that it has the amino acid sequence ID N2.

6. A method of obtaining a precursor polypeptide having SCCE activity, characterized in that the method includes the following stages: a) the ktoroy, obtained in stage (a), (b) cultivating the host organism, obtained in stage (b), under conditions suitable for expression of the precursor polypeptide, g) collecting the precursor polypeptide.

7. Pharmaceutical composition for treating disorders associated with keratinization, characterized in that the composition includes native or recombinant chymotrypsinlike enzyme corneal layer (SCCE) in an amount of from 0.001 to 25 wt.% from the total mass of the composition.

 

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