Polypeptide synthesis method (variants), dna (options), vector (options), cell, applying a polypeptide, a pharmaceutical composition (options)

 

(57) Abstract:

The invention relates to genetic engineering and medicine, in particular to a new polypeptide. The polypeptide inhibits the binding of TGF-alpha (growth factor T-cell) with its receptors. Amino acid sequence is shown in the description. Adsorb urine at acid pH on the kaolin. Extracted it with ammonia. Elute the fractions obtained on the resin Bio-Rex 70 using ammonia. Obtained after elution fractions blueroot on DEAE-Sepharose resin using acetate buffer, then CM-Sepharose resin using acetate buffer, then the resin 18 HPLC (HPLC) using a mixture of acetate buffer and acetonitrile, and then the resin D-Zephyr using acetate buffer, then the resin 18 HPLC (HPLC) using a mixture of aqueous trichloroacetic acid and acetonitrile. Then, on the resin D-Zephyr using acetate buffer. Get polypeptide or containing its basically pure protein. The polypeptide is encoded by DNA nucleotide sequence listed in the application materials. Expressing the vector comprises a DNA fragment encoding the polypeptide. The strain of E. coli transformed expressing vector and produces poly is the formation of prokaryotic or eukaryotic cells, transformed expressing vector and select it from the culture medium. Farmcampsite that has the property to inhibit the binding of TGF-alpha, contains a therapeutically acceptable amount of a polypeptide or mostly pure protein in combination with one or more therapeutically acceptable drives or diluents. Drug having anti-inflammatory and/or antikoaguliruyuschey, and/or antitumor activity comprises a polypeptide with the amino acid sequence listed in the application materials. The invention allows to obtain a new protein with a relatively low molecular weight, which can be used to obtain anti-inflammatory, antikoaguliruyuschee treatments, and also means possessing antitumor activity. 19 C. and 1 C.p. f-crystals, 8 ill.

The invention relates to a new protein, named Component B. More specifically, the present invention relates to a new protein, derived from the urine; to obtain this protein from the urine; its synthesis by techniques of recombinant DNA using genomic DNA or cDNA copies specified new protein; and farmacevticheskom relative low molecular mass polypeptide and nature, and can be selected in the process of extraction and purification derivatives of urine. To do this, the urine of a person treated with absorbent materials such as kaolin, then subjected to filtration, ion exchange chromatography and high resolution chromatography, preferably in accordance with the following equipment; and after lyophilization get a connection in the form of a white amorphous powder, which in reversed-phase liquid chromatography high pressure (PF-ghvd) moves as a single peak and which has a molecular mass of about 9 kDa, as shown by electrophoresis in polyacrylamide gel with dodecylsulfate sodium (LTOs-page) performed in reducing conditions. This protein was named Component B.

Amino acid sequence of the Component B is presented below as SEQ ID No. 1 (the list of sequences, see the end of the description).

The present invention relates to the production of a new protein, named Component B, the method comprising the allocation of crude fractions of the compounds of cialisovernight concentrate urine after processing adsorbing agent and purification using ion exchange chromatography and high resolution chromatography, as opielka it exists in large quantities, and therefore can be used in industrial production. More specifically, the present invention relates to a polypeptide having the sequence SEQ ID No. 1 and its salts, functional derivatives, precursors and active fractions and their active mutants, i.e., other proteins or polypeptides in which one or more amino acids have been deleted, substituted by other amino acids or added in order to obtain polypeptides or proteins having activity similar to the activity of Component B; in addition, the present invention also relates to hybrid proteins, i.e., polypeptides containing the Component B or mutation, legirovannye with another protein, and having a longer half-life in the physiological fluids in the body. Therefore, the Component can be subjected to fusion with another protein such as an immunoglobulin.

The term "salt" used in the present description, means salts of carboxyl groups and salts of functional amino groups of the compounds synthesized by known methods.

Salts of carboxyl groups can be inorganic salts, such as salts of sodium, potassium and calcium, as well as salts formed redstavlyaete a salt, formed with inorganic acids such as hydrochloric acid, and organic acids such as acetic acid. The term "functional derivative" used in the present description, refers to derivatives, which can be obtained from the Functional groups present in side chains of amino acid residues or at the end of the N - or C-groups, in accordance with known methods; and which are included in the scope of the present invention in the case, if they are pharmaceutically acceptable, i.e. have activity similar to the activity of the protein, or give pharmaceutical compositions containing these derivatives, toxic properties.

Examples of such derivatives are, for example, esters or aliphatic amides of carboxylic groups, and N-acyl derivatives of free amino groups, O-acyl derivatives of free hydroxyl groups, and derivatives formed by acyl groups, for example alkanolamine or rollname groups.

The term "precursor" means a compound that the body of man or animal turn into Component B. the Term "active fractions" of the protein of the present invention means any who are connected with him related molecules or residues for example, residues of sugars or phosphates, or aggregates of the polypeptide molecule, provided that such fragments or precursors, as drugs have activity similar to the activity of Component B.

The present invention also relates to mixtures of polypeptides and derivatives, as defined above.

In the second version of my implementation of the present invention relates to a method for producing a Component B, which provide a crude fraction from cialisovernight concentrate urine, after it is processed adsorbing agent and purification using ion exchange chromatography and high-resolution chromatography.

Component B preferably produced by the method illustrated in Fig. 1 and includes the following stages:

a) adsorption of urine at acid pH on the kaolin and its extraction with ammonia;

b) elution of fraction (a) in the resin Bio-Rex 70 using ammonia;

c) elution of fraction (b) on DEAE-Sepharose resin using acetate buffer;

(d) elution of fraction (c) CM-Sepharose resin using acetate buffer;

e) elution of fraction (d) on C18-resin (HPLC) using a mixture of acetate buffer and the s fraction (f) on D-Zhephyr-resin using acetate buffer;

h) elution of fraction (g) on C18-resin (HPLC) using a mixture of water triperoxonane acid and acetonitrile;

i) elution of fraction (h) on D-Zhephyr-resin using acetate buffer.

In addition, the present invention relates to recombinant DNA molecules that contain nucleotide sequence encoding the polypeptide of the present invention, the active mutant or hybrid proteins; and to the containing expression vectors; cell host transformed by these vectors; and the method of production of these polypeptides, their active mutant or hybrid proteins by culturing these transformed cells in an appropriate culture medium. The term "recombinant DNA molecule" refers to genomic DNA, cDNA, synthetic DNA and combinations. In particular, the present invention relates to the nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

SEQ ID No. 2 is a genomic DNA sequence, encoding a Component B.

In Fig. 2 shows the restriction map of the transcriptional units of Component B.

SEQ ID No. 3 is a cDNA-posledovatelno B, showing restriction sites. Cloning of the Component B can be implemented in different ways. In one of these ways get oligonucleotide, or a mixture of nucleotides, the sequence of which is driven shoda from the sequence of the Component B or its fragment used as a probe for cloning cDNA or genomic DNA that encodes a Component B.

SEQ ID No. 4 represents the amino acid sequence encoded by the genomic DNA (SEQ ID No. 2) and cDNA (SEQ ID No. 3).

The present invention also relates to recombinant DNA molecules which hybridize with the DNA sequence that encodes a Component B or its fragment.

The desired gene can contain or not contain natural introns, and may be obtained, for example, by extraction from the respective cells, followed by purification with known methods. Related products DNA, human genomic DNA is cut by suitable means, preferably by using restrictively enzymes and the resulting fragments injected into appropriate recombinant vectors to create a library of DNA. These vectors can be selected using syntheti the invention.

In accordance with the present invention, genomic DNA Component B is isolated and clone.

On the other hand, cells expressing the Component B may be selected corresponding mRNA and used for producing complentary DNA (cDNA) with known methods. This cDNA after transformation in the double helix can be introduced into an appropriate vector, which is then used for transformation of a suitable host cell. After this culture are selected using the corresponding probe in order to obtain a cDNA encoding the target sequence. After selection of the desired cDNA clone is subjected to basically the same manipulations that and genomic DNA.

cDNA does not contain introns. Due to the degeneracy of the genetic code, can be used in different codons that encode a particular amino acid, so that producyrovtsa one or more oligonucleotides, each of which is able to encode fragments of Component B. However, only one member of the pool has a nucleotide sequence identical to the sequence of this gene. The presence of this member in the pool and its ability to hybridisierung with DNA in the presence of other members of the pool of postures which should be used for cloning of the gene encoding the target peptide. Alternatively, a single nucleotide containing a sequence about which can theoretically with the highest degree of probability that it is able to encode gene fragments of Component B (as described in "rules for the use of codons" (rules for the use of codons") - Lathe R. et al., Molec. Bid., 183:1-12 (1985)), allows the identification of complementary DNA encoding the Component B or its fragment.

Methods of hybridization of nucleic acids are known and described in the literature (see , for example, T. Maniatis et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1982); B. T. Haymes et al. , Nucleic Acid Hybridization: A Practical Approach, LRL Press, Oxford, England (1985)). Hybridization using a specific probe or group of nucleotide probes allows to identify in a genomic or cDNA library with a DNA sequence capable of such hybridization, which, after further analysis, was confirmed by its ability to encode a polypeptide of the present invention (i.e., Component B). Oligonucleotide containing such a complementary sequence can be synthesized and used as probes for identification and isolation of the gene for the polypeptide of the present from the specific to the Component B, will be selected in accordance with the above method, it can be synthesized and hybridized to DNA or preferably with cDNA derived from the cells that can Express the desired gene, preferably after enrichment of the source of the desired cDNA sequences, for example by extraction of RNA from cells that produce high levels of the desired gene, and the subsequent transformation of this RNA in the corresponding cDNA using reverse transcriptase.

Alternatively, it may be suitable synthesized oligonucleotides specific for the Component B, and then used as primers to amplify cDNA fragments of the Component B with PACE-PCR (M. A. Innit et al. PCR Protocols A Guide to Methods and Application, Academic Press, 1990).

In particular, in accordance with the present invention is first to identify the most appropriate source for mRNA Component B was carried out search for the relevant human tissues and cell lines. This was skanirovali human tissues originating from brain, kidney, liver, lung, heart, pancreas, placenta, spleen, testis, thymus, and uterus, as well as cell lines epithelioid carcinoma, promyelocytic leukemia, adenocor the analysis, based on the polymerase chain reaction using reverse transcriptase (RT-PCR).

The best source of mRNA was tissue of the uterus of the person.

From this tissue using the method of rapid amplification, called "fast 3'- and 5'-amplification of cDNA-ends", received cDNA-Lona Component B.

Then a DNA molecule encoding the B Component and obtained by the above method, introduced in expressing vectors using known techniques (Maniatis and others, see above). Double helix cDNA are ligated into plasmid vectors using, for example, a method that uses synthetic DNA adapter, or using a method which is "binding on blunt ends".

For expression of the target protein expressing vector should also include specific nucleotide sequence containing information necessary for the regulation of transcription and translation DNA that encodes a desired protein, so that could be the expression of the desired gene and the production of protein. First, to ensure transcription of the desired gene, this gene should be placed promoter, which can be recognized by the RNA-polymer of known promoters, "working" with varying degrees of effectiveness (strong promoters and weak promoters), are divided into promoters used in prokaryotic cells, and the promoters used in eukaryotic cells.

The promoters used in the present invention can be constitutive (unregulated) promoters such as the promoter int lambda bacteriophage promoter B1a gene-lactamase in pBR322, and the CAT promoter of the chloramphenicol-acetyltransferase pPR325, etc. or inducible promoters, such as promoters prokaryotes, for example the right and left main promoters of lambda bacteriophage (Pl and Pr), the trp promoters, rec A, lac Z, lac I, ompF and gal E. coli or hybrid promoter, a trp - lac, etc. (Glic B. R., J. Ind. Environ., 1:277-282 (1987)).

To ensure efficient translation of mRNA is necessary that along with strong promoters capable of producing very large quantities of mRNA, providing high levels of gene expression in prokaryotic cells, used as binding sites with ribosomes. As example can serve as a sequence Shine-Dalgano (SD) placed at an appropriate distance from the initiating codon.

For eukaryotic cells, the dependence on the nature of a particular owner.

These sequences can occur from viral sources, such as adenovirus, papilloma virus, simian virus, or so forth, where the signals of regulation are associated with specific gene with high expression level. As examples may serve the TK promoter of herpes virus, the promoter of the virus SV 40 promoter gene gal4 yeast, etc. Signals of transcription initiation can be selected so that the induction of suppression or activation could appropriately modulate gene expression.

Then a DNA molecule containing a nucleotide sequence encoding a Component B of the present invention, together with signal sequences that regulate transcription and translation, is introduced into the vector, which is able to integrate the sequence of the target gene in the chromosome of the host cell.

Cells carrying in their chromosomes introduced DNA can be selected by introducing one or more markers, allowing you to select the cells that contain expressing vector. Such markers can tell the cells, for example, resistance to antibiotics or heavy metals (such as copper). Selective gene may be the right near St the Amu cell by constructible. To implement a higher level of gene expression, it may be necessary also the presence of other elements. These elements can be, for example, enhancers, transcription signals termination of transcription and introns. Expressing the vectors which contain such elements are described in Okayama, H., Mol. Cell Biol. 3: 280 (1983).

When selecting a particular plasmid or viral vector must be taken into account the following factors: ease of detection of cells containing the vector, i.e., these cells should be easily separated from cells that do not contain the vector; the number of copies of the vectors that you want to get in this particular host; and the possibility or impossibility of the transfer vector among different host cells.

Preferred prokaryotic vectors include plasmids that can replicate in E. coli, such as BR322, CoIEI pSC101, pACYC 184, etc., (Maniatis T., and others, see above), plasmids of Bacillus, such as pC194, pC221, pT127, etc., (Gryczan, T. M. The Molecular Biology of the Bacilli, Academic Press, NY, 307-329 (1982)), plasmids, such as PIY 101 (Kendall, K. J. et al., J. Bacteriol. 169:4177-83), plasmids Peseudomonas (John J. F. et al., Rev. Infect. Dis. 8: 693-704 (1986)) (Izaki, K. Jpn. J. Bacteriol. 33: 729-742).

Preferred eukaryotic vectors D. et al., Miami Wint Symp. 19: 265-274) (Broach J. R. The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor, NY, 455-470 (1981) (J. R. Broach, Cell 28: 203-204 (1982)) (Bollon D. P. et al., J. Clin. Hematol. Oncol. 10: 39-48 (1980)) (Maniatis T. Cell Biology: A Comprehensive Treatise vol. 3: Gene Expression, Acad. Press, NY, 563-608 (1980)).

Received expressing the vector is introduced into an appropriate host cell by standard techniques, such as transformation, transfection, lipofection, conjugation, fusion of protoplasts, electroporation, precipitation of calcium phosphate, direct microinjection, etc. for the purposes of the present invention can be used prokaryotic or eukaryotic cell hosts.

The preferred prokaryotes are bacteria, such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.

Especially preferred is E. coli, such as strain 294 E. coli K12 (ATCC 314446), or E. coli X1776 (ATCC 31537), E. coli W3110 (F -, lambda, ATCC 27325).

Preferred eukaryotic cells hosts are mammalian cells such as human cells, monkey, mouse, or hamster (Cho cells Chinese hamster ovary), because these cells provide post-translational modification of protein molecules, for example, the correct installation and glycosylation in the right positions.

Weia various techniques of recombinant DNA, which uses a sequence of strong promoters and a large number of copies of plasmids, the desired protein can be produced in yeast.

After the introduction of the vector into the host cell these cells are cultivated in an environment conducive to selective growth of cells that contain the vector.

Expression of the cloned DNA sequence enables generation of the Component B, it is mutant or fragment. Produced thus secrete protein or purified by traditional methods, for example by extraction, precipitation, chromatography, electrophoresis, etc., or by affinity chromatography using antibodies against a Component B, immobilized on a column of gel. Component B may also be produced as a protein, the secretory of the milk of transgenic mammals.

In yet another embodiment, the present invention relates to the use of Component B, its salts, functional derivatives, precursors or active fractions as a medicine.

In particular, Component B has anti-inflammatory, anticholergenic and antitumor properties. In addition, Component B may be used on the and hormonal disorders antigenes etc.

In practice, it has been shown that the Component B inhibits the binding of TGF-alpha to its receptor with an affinity constant, component Ki= 0,7710-10M, and measured by the displacement of the I125- TGF-alpha to its receptor, derived from the cell membrane to A 431.

The present invention also relates to pharmaceutical compositions containing a therapeutically active amount of the Component B in combination with pharmaceutically acceptable excipients or diluents. Such compositions can be obtained for oral, rectal, intranasal, and preferably for parenteral administration.

In addition, in accordance with the present invention, the Component B can be used for local application.

Compositions of the present invention can be also manufactured in the form of slow release, for example in the form of subcutaneous implants based on liposomes or microcapsules copolymers of lactic and glycolic acids.

Other embodiments of the present invention will be apparent from the following detailed description of the invention with specific examples.

Example 1

a) stage 1

For source material, representing the urine of a person, was added hydrochloric acid until then, until the pH does not become equal to 3.0. After desantirovaniya to urine sediment (10 g/1 l of original urine) was added to the kaolin.

The resulting suspension was left for 16 hours, and then centrifuged.

After removal of the supernatant kaolin were extracted with 2 M ammonia at pH= 11.0 in.

the pH of the ammonia eluate was brought to 8.0 and concentrated using an ultrafiltration membrane (cut 1000 Da). The whole procedure was carried out at a temperature of 4oC.

b) stage 2

To a solution of stage (a) was added acetic acid until then, until the pH does not become equal to 4.0, and then added to the resin Bio-Rex 70, pre-equilibrated in acetate buffer at pH=4,0.

The resulting solution was left to stir for 4 hours and then filtered on a filter press.

The adsorbed material was suirable from the resin Bio-Rex 70 using ammonia at pH=9,0.

Chromatographic eluate was concentrated using an ultrafiltration membrane (cut 1000 Yes).

The whole procedure was carried out at a temperature of 4oC.

c) stage 3

MA Is Separate, pre-equilibrated at pH=5,6.

After adsorption, elution was carried out using ammonium acetate buffer 0.5 M at pH=5,6. Then chromatographic eluate was concentrated using an ultrafiltration membrane (cut 1000 Da). The whole procedure was performed at 4oC.

d) stage 4

Material stage (c) balanced in acetate buffer at pH=4.5 and adsorbing on ion-exchange resin similar to the CM-Sepharose and pre-equilibrated at pH=4.5.

After adsorption, elution was carried out by ammonium acetate buffer of 0.15 M at pH = 4.5. Chromatographic eluate was concentrated using an ultrafiltration membrane (cut 1000 Da). The whole procedure was carried out at a temperature of 4oC.

e) stage 5

Material stage (d) was purified at a temperature of 25oC using reversed-phase liquid chromatography high resolution on the C18 resin, equilibrated in ammonium acetate buffer 0.05 M at pH = 5,6.

The adsorbed material was suirable from the resin using a solution of ammonium acetate containing 30% (vol./about.) acetonitrile.

Chromatographic eluate was concentrated by distillation (40oC) in the DE-52 and equilibrated in ammonium acetate buffer 0.02 M at pH=5,6.

Elution of adsorbed material was carried out using 0.25 M buffer. Concentration was carried out using an ultrafiltration membrane (cut 1000 Da). The whole procedure was carried out at a temperature of 4oC.

(g) stage 7

Material stage (f) was purified on a column of pre-Packed ion exchange resin, such D-zephyr (utverzhdenii with Sepracor), and equilibrated at pH= 6.2 in 20 mm buffer solution of sodium acetate (buffer A).

Elution of the absorbed material was carried out with a gradient from 100% buffer A to 100% buffer solution of sodium acetate containing 1 M NaCl (pH=6,2).

(h) stage 8

Material stage (g) was purified using reverse-phase liquid chromatography high resolution on the C18 resin at a temperature of 25oC.

After adsorption, elution was carried out with a linear gradient mixture of an aqueous solution of 0.1% triperoxonane acid and acetonitrile acidified with 0.1% triperoxonane acid.

Chromatographic eluate was concentrated by distillation (45oC) in vacuum and liofilizovane.

i) stage 9

Repeated stage 7. The result of this procedure was given final pradeonent B

In order to determine the main physical-chemical characteristics of the Component B obtained purified material from human urine were subjected to the following analytical processing.

a) Amino acid sequence

Amino acid sequence of the Component B was determined in accordance with the method of Edman.

The analysis was performed using sequencing machine Applied Biosystem, model 477A in accordance with the manufacturer's instructions.

The specified analysis gives the possibility to identify the amino acid sequence of Component B with 81 amino acid residues described in SEQ ID No. 1.

b) Determination of molecular weight

The analysis was performed using mass spectroscopy electron impact (MS-ED), in the result, it was found that molecular weight is 8937,9 Yes. This analysis revealed five disulfide bridges and the rest 80 Yes, belonging to the group of SO4associated with Tyr (39).

Example 3

The allocation of genomic DNA Component B man

A library of human genomic DNA in the lambda-step vector EMBL-3 SP6/T7 was supplied by the company Clontech (cat. N HL 1067, J. Lot N 1221). Genomic DNA was extracted from human placenta and partially digested FA sucrose to obtain fragments ranging in size from 8 to 22 Kb.

Media for cultivation

Cells of E. coli K802 supplied by the company Clontech (cat. N C1004-1) was cultured in LB medium, supplemented with 10 mm MgSO4and 0.2% maltose (medium for cultivation).

Phage library was diluted in 0.1 M NaCl, 8 mm MgSO4, 50 mm Tris-Cl (pH 7.5) and 0.01% gelatin (SM).

A DNA library was cultured on LB-plates with 1.5% agar. The top agar for cultivation library contained 0,136 M NaCl and 0.6% agarose and 1% tripton (Merck cat. N 7213).

Reagents for hybridization

20 x SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.

Hybridizers solution of 5 x SSC, 0.02% of SDS, 0.1% of N-lauroylsarcosine, 0,5 blocking reagent (Boehringer cat. N 1096176).

Solution A for washing (HRP-oligonucleotide) - 3 x SSC, 0,1% SDS, urea, at various concentrations, depending on the type of probe (see below).

Solution B for washing (32P-oligo CBEX4L) - 1 x SSC, 0,1% SDS.

The detection system

Hybrids of the HRP-oligo/DNA was detected using the ECL-kit and exposed to film (Hyperfilm ECL from the company Amersham, cat. N PPM 2106 and 2104, respectively).

32P-Oleksandrivna filters were discovered by eksponirovanie film-max (Hyperfilm-max, Amersham, cat. N PRN10).

Oligonucleotides

Oligonucleotides with sitesyou of OPC cartridges (Applied Biosystem cat. N 400771) or by electrophoresis in denaturing Polyarylamide gel (SDS page).

Oligonucleotides CB1, CB2 and CBEX2L used as probes modified with aminoadipate MMT-C12 (Clontech cat. N 5206-1) in the procedure of the last cycle of synthesis.

Horseradish peroxidase (HRP, Boehringer cat. N 814393) conjugatively with modified oligonucleotide in accordance with the description of M. S. Urdea (Nuc. Ac. Res. 16, 4937, 1988), using 1,4-phenylenedimethylene (Aldrich, cat. N 25855-5) as homobifunctional cross-linker.

HRP-Oligonucleotide probes were purified using anion exchange chromatography high resolution (VIHR) on a column with Nucleopac PA-100 (Dionex cat. N 043010). Then elution was carried out 20 mm buffer solution of sodium phosphate at pH = 6.0 and a linear gradient of sodium chloride (0.2 M to 1.0 M) for 30 minutes.

Purified HRP-oligonucleotides were concentrated using Method 10, washed with phosphate buffer solution (PBS) and stored in the dark at 4oC.

The concentration of HRP-oligonulceotide figured if OP403(403= 89.5 cm-1mm-1).

Then synthesised oligonucleotides, listed at the end of the description.

Titration library

Genomic DNA billionaire 0.3 ml cell culture of E. coli K802, seasoned throughout the night, various dilutions of the library, ranging from 2 to 10-3up to 2 10-7. The mixture of cells of the library were incubated at room temperature for 20 minutes, and then within 10 minutes, the mixture was incubated at 37oC. Infected cells were mixed with 4 ml of top agarose, pre-heated at a temperature of 50oC, and then poured into 10-inch tablets with agar, preheated at 37oC. These plates were incubated overnight at 37 ° oC.

The number of plaques was counted in each tablet. Tablets with duplicate cultures were obtained for each of the titer of the library. As expected, the titer genomic DNA library was 5 109PFU/ml.

Screening of the library

Cells of E. coli K802 were cultured over night at 37 ° oC., 0.6 ml of cell culture was infected by aliquot of the library (6 of 104The COMBAT), suspended in CM. Infection and cultivation was carried out as described above, except that used 9 ml of top agarose and 15-inch tablets.

Polyethlyene plaques were transferred to nylon membrane Hybond N+(Amersham) in soothill colonies uppermost filter paper soaked in 1.5 M NaCl and 0.5 M sodium hydroxide for 7 minutes.

Then blokirovanie DNA was neutralized by applying filters on a filter paper soaked in neutralizing solution (1.5 M NaCl, 0.5 M Tris-Cl (pH of 7.2), 1 mm EDTA)) (in each case, 2 x 3 min).

Filters were washed in 2 x SSC and dried by air. DNA was fixed on the membrane by applying filters on a filter paper immersed in 0.4 M NaOH for 20 minutes.

Then the filters were washed in 5 x SSC for one minute, and then kept in a plastic bag at 4oC up to hybridization. Human genomic DNA library (1 of 106clones) were skanirovali with high density using oligonucleotide probes HRP-CB2. Were then selected 20 positive clones.

Six positive clones was re skanirovali using oligonucleotide probe HRP-CB1 and oligonucleotide probe HRP-CB2. Three clones, designated 4D, 12B and 15, were confirmed as positive for the gene of component B.

Hybridization

The filters are pre-incubated for 30 minutes at 42oC hybridizers solution, and then was hybridisable using suitable theoC for 45 minutes, and finally washed twice (15 min each) at a temperature of 42oC in wash solution containing urea in the appropriate concentration (see below).

Then the filters were washed in 2 x SSC at room temperature and hybridized plaques were detected using ECL reagents and exposed to film Hyperfilm within 60 minutes.

To minimize nonspecific hybridization with E. coli and DNA lambda phage were experimentally determined conditions for washing HRP-oligodendrogenic filters. Serial dilution ranging from 500 to 15 attomole target DNA were marked spots on the membrane Hybond N+in the presence of DNA of lambda phage (10 ng). DNA of lambda phage DNA and E. coli (10 ng each) were used as negative control. Thereafter received several strips and used them in experiments for hybridization using 5 ng/ml oligonucleotide probe. The leaching was carried out using a wash solution A, containing 0%, 9%, 18%, 27% and 36% urea.

18% and 27% urea was effective for CB1 and CB2, respectively. Filters, hybridized using CBEX2L, the flushing solution A, containing 18% mcevenue washed in wash solution B at a temperature of 45oC.

Subscriberbase plaques

Positive colonies were collected in a Pasteur pipette and transferred into a vessel containing 1 ml of CM + 1 drop of chloroform. After 2-hour incubation with shaking at room temperature, the phage suspension was stored at 4oC.

Breeding 10-3the phage suspension was applied to a 10-inch Board and riscrivibili two duplicate filters with two oligoside, i.e., the one used in the first screening, and the other corresponds to the adjacent area of component B.

Independent clones positive with both probes were selected and resuspendable as indicated above.

Preparation of phage drains

Positive clones were propagated by infection of cells of E. coli K802 and growing at 15-cm agar boards. After incubation in the cut ON at 37oC merging the lysate was collected from agar plates with 10 ml of SM. Were added a few drops of chloroform, and the cell debris removed by centrifugation at 3000 rpm for 5 min at

4oC and the clear supernatant containing the phage was transferred in 50% glycerol, bottled and frozen at -80oC.

Extraction ragovoy DNA

2 109the culture medium ON at 37oC. At the end of the incubation was carried out a complete lysis of the cells by adding to the culture of chloroform 5 ál/ml

Phage DNA was extracted using Quiagen per manufacturer's instructions.

Sequencing ragovoy DNA

Phage DNA was sequenced using cyclic securitysage kit from Applied Biosystem (cat. N 401388) with an automated DNA sequencing machine (Applied Biosystem mod. 373A). The phage DNA was extracted from clones 4D, 12B and 15, and sequenced loops.

Primers for sequencing were selected either on the basis of the amino acid sequence of Component B (CBF1, CBF2, CBP1, CBP2), or based on data from sequencing of cDNA or genomic DNA. Data sequencing indicated the three clone containing the full length gene of Component B.

Analysis of restriction ragovoy DNA

Phage DNA was subjected to single or multiple digestion restriction enzyme. DNA fragments were separated by electrophoresis on a 0.6% agarose gel, and then blokirovala on the membrane Hybond N+. Filters re-probed using oligonucleotides CBEX2L, CB2, and CBEX4L corresponding to exons 1, 2 and 3, respectively.

Subclavian Component B gene in pBluescript 11SK

Restriction any using oligonucleotide probes, specific to the three exons of the Component B has detected the presence of the full gene Component B containing a 5.2 Kb EcoRI-enzyme (Fig. 5).

Phage DNA was extracted from clone 4D and digested with the enzyme EcoRI. The resulting DNA fragments were separated by electrophoresis on agarose gel. 5.2 Kb fragment was purified using Quaex (qiaoen cat. N 20020) and ligated into EcoRI-linearized pBluescript 11 KC (Stratagene cat. N 212207). The E. coli strain XLI-Blue (Stratagene cat. N 200268) transformed using ligiously mixture, and transformirovaniya cells were selected on Ap/Tc-cups. As shown by restriction analysis using enzyme EcoRI, was selected a single clone containing the desired plasmid, which was named pBS CB4D.

Then was carried out by restriction analysis using SmaI, kpni restriction sites, HindIII, SfiI, AccI, NotI, SalI, XhoI, EcoRI, ClaI, HincII, HindII, ScaI, BglII, Aat2, NcoI, NheI, HpaI and MluI. In addition, performed southern blot analysis using oligonucleotide probes specific for the Component B and the plasmid pBS CB4D after single and double digestion.

In Fig. 6 shows the restriction map of the plasmid pBSCB4D.

In Fig. 4 shows the restriction map of the gene of Component B. Component B Gene contains 3 exons separated from each other by the two introns is tidow netransliruemoi mRNA and the sequence encoding the 19 amino acids of the proposed signal peptide. This exon is separated from exon 2 intron, whose length is 410 p. O.

Exon 2, having a length of 120 p. O., 3 encodes amino acids of the proposed signal peptide and 37 amino acids of the Mature protein. This exon is separated from exon 3 intron, whose length is about 550 p. O.

Exon 3, having a length of 326 p. O., encodes the C-terminal 44 amino acids of the Component B and contains 192 nucleotide noncoding mRNA, which has a polyadenylation signal (TATAAA), located at a distance of 14 p. O. ("upstream") from the site 3'-processing, the end of which is added polyA-tail.

In particular, it was found that in three genomic clones sequence encoding a signal peptide contains a Leu codon in position II of the proposed signal peptide.

It was found that the amino acid sequence of the Component B produced genomic gene, identical sequences, experimentally defined by the splitting method for Admino.

Sequence analysis of the property before exon 1 revealed promoter region (Fig. 3) containing a TATA-box (b - 28), and various, they are situated a few E-blocks.

TATA-block is the preferred binding site for transcription initiation factor TFIII. GC-rich site is a binding site for sp-1, the main transcription factor involved in transcription of a wide range of genes (reduced and Splicing B. D. Hames & D. M. Clover, Eds. , IRL Press, 1988).

The AP-1 site is a binding site for AP-1 complex, transcription factor formed by c-fos and c-jun. The AP-1 site present in several genes responsible for growth and differentiation of cells. AP-1 is one of several cis-elements mediating the response in relation to activators of protein kinase C (The hormonal control of gene reduced P. Cohen & J. G. Foulkes End. Elsevier, 1991).

AP-2 site is a target for AP-2 transcription factor, activated by PMA and cAMP (ibidem).

E-blocks are generalized sequences found in multiple enhancer regions and play an important role in determining tissue-specific gene expression. E-the block contains a sequence CANNTG, where two bases located in the middle, can vary for each specific E-block. (R. E. Kingston Current Opinion Cell Biol. 1989; 1, 1081-1087).

The promoter Component B contains potential sensitive element to the receptor is corticoide.

Subclavian Component B gene in the vector for expression in mammalian cells

It is known that the production of rec-protein in mammalian cells can be increased by the presence of introns. Genomic DNA Component B can be expressed in mammalian cells.

To do this, 1364 p. O. - fragment extending from +50 to +1413 Component B gene, was cut out from plasmid pBSCB4D by digesting enzymes PvuII and NarI. In Fig. 2 shows the restriction map of transcription Component B, where PvuII and NarI sites are situated at the ends. Full Component B gene was restored by ligating this fragment with a synthetic oligonucleotide, reproducing the 5'-end of the gene, flanked by appropriate restriction site for subsequent cloning of the gene in eukaryotic expressing plasmid.

Example 4

The selection of cDNA clones Component B

To obtain a partial cDNA clones corresponding to the 5'- and 3'-ends of the mRNA Component B used method for rapid amplification of cDNA-ends described by Frohman et al. (1988) Proc. Natl. Acad. Sci., USA 85; 8998. Partial clones containing overlapping DNA sequence, and therefore they can be used to construct the curtain of Fig. 7.

3'-PAGE DNA sequence of the second exon of the gene of the Component B used to design gene specific primers CKCB1 (5'-TCAACTCCTACACCTCCAACCAC-3') (SEQ ID No. 21). The cDNA synthesis was initiated from A poly-end poly (A+RNA uterus person using as primers the oligonucleotide 5-CGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3' (SEQ ID N 24), called adapternum primer AP. cDNA was used as template for polymerase chain reaction (PCR), where the primers used CKCB1 and AP, which has produced a fragment with approximately 450 p. O. and the corresponding 3'-end cDNA of Component B.

5'-PAGE, DNA sequence 450 p. O. 3'-PAGE-fragment designed primer CKCB7 (5'-CGTCAGAGAGGAGGTC-3) (SEQ ID No. 22), which was used as a primer for synthesis of cDNA from poly (A+RNA uterus person. After cleaning in order to remove mRNA and primers CKCB7 end oligodeoxythymidine was added to the 3'-end of cDNA. This cDNA with the "tail" was used as template in a PCR, primers served as "nested" primer CKCB2 (5'-ACCGTCACCAGCGTGGTC-3') (SEQ ID N 23) and the anchor primer (ACP, 5'-CTACTACTACTAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') (SEQ ID No. 25), or a mixture of ACP and universal primer amplification (UAP, 5'-CTACTACTACTAGGCCACGCGTCGACTAGTAC-3') (SEQ ID No. 26). CKCB2 was hybridisierung is the result of the received fragment of approximately 230 p. O., which contained the DNA sequence corresponding to the 5'-end of mRNA of Component B.

Using the General scheme of the experiment (for example, polyacrylamide gel electrophoresis, precipitation with ethanol, ligation, and digestion restricteduse endonucleases), bacterial culture medium (e.g., LB) and chemicals (e.g. phenol) is described Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, unless specifically indicated.

Procedure 3' RACE clone

3' RACE system for rapid amplification of cDNA ends was purchased from Life Technologies, Inc., Grand Island, NY, Poly+-RNA uterus person purchased from Clontech Laboratories, Inc., Palo Alto, CA. The synthesis of the first cDNA strands was performed using the Protocol and reagents supplied with 3'-RACE system. To do this, 1 μl (1 μg) of poly A+-Uterine RNA was combined with 1 ikl 10 μm solution of AP and 12 μl of water, treated with diethylpyrocarbonate (DEPC), and the resulting mixture was heated for 10 minutes to 65oC. After cooling the mixture on ice, the reaction components were added so that the final composition had approximately the following composition: 20 mm Tris-HCl (pH of 8.4, 50 mm KCl, 2.5 mm KCl, 2.5 mm MgCl2, 100 μg-ml bovine serum albumin, 500 nm AP, 500 μm caidahl (20%) of the reverse transcriptase superScript. After a 30 minute incubation at 42oC the reaction mixture was cooled on ice, and added 1 μl (2 units) RNase H. Then there was Mcasa N-digestion for 10 minutes at 42oC. the reaction mixture is kept at -20oC until the implementation of the PCR.

For the implementation of PCR were obtained four identical mixture (40 μl), each of which had the following composition: 1 μl poly A+to the DNA of the uterus in 40 mm KCl, 70 mm Tris-HCl (pH 8,8) 0.1% Triton X-100, 1 mm MgCl2, 0.25 μm CKCB1, and 0.5 µm AP. The reagents of the 3'-RACE system was not used for PCR. CKCB1 - and AP-primers were synthesized on an oligonucleotide synthesizer model 392 (Applied Biosystems, Inc.). After unlocking lyophilization and resuspendable in DEPC-treated water was measured optical density of each solution at a wavelength of 260 nm. Based on the measurements of optical density, in DEPC-treated water to prepare a 10 μm solution of each of the oligonucleotides. The crude oligonucleotide solution was used for PCR reaction without additional purification. The concentration of crude AP, which gave results identical to the results obtained using the 3' RACE system was determined experimentally: 0.4 µm untreated AP equivalent to 0.2 μm AP, Zack is URS 94oC, and then each mixture was added 10 μl of a mixture containing the following components: a 1.25 units of DNA polymerase AmpliTag (Perkin Elmer Cetus, Norwalk, CT), 40 mm KCl, 70 mm Tris-HCl (pH 8,8), 0.1% Triton-X-100, 1 mm MgCl2and 1 mm each of dATP, dTTP, dGTP and dCTP. The final concentration of each reagent in the PCP was approximately 1 μl of cDNA uterine 50 µl of 1.25 units of DNA polymerase AmpliTag 50 μl; 40 mm KCl, 70 mm Tris-HCl (pH 8,8), 0.1% of Triton X-100, 1 mm MgCl2, 0.2 μm CKCB1, 0.4 µm AP and 0.2 of each of dATP, dTTP, dGTP and dCTP. After 5-minute incubation at 94oC, carried out PCR thermal Cycling "Touchdown" in accordance with the description Don R. H., Cox, P. T., Wainwright, B. J., Baker, K., & Mattick, J. S. (1991) Nucl. Acids Res. 19, 4008, by varying the annealing temperature from 73oC to 63oC.

After PCR-amplificatio four reaction mixtures were pooled, and the DNA products were fractionally by size by electrophoresis on 5% polyacrylamide gel. DNA product having a length of about 450 p. O., cut out from the gel and purified by electroelution in the dialysis tube. The eluate was extracted with a mixture of phenol and chloroform (50:50, vol/vol.), precipitated with ethanol, dried off and was respondible in 10 ál of sterile water.

Due to the independent matrix end transferase activity of DNA on the Lae D. P. (1989) Nucl. Acids Res. 17, 3319), purified PCR fragment of 450 p. O., had, as expected, one deoxyadenosines the remainder of each 3'-end. For sublimirovanny and characterization of PCR-fragment was obtained T-vector pBluescriptsk +" (Stratagene, La jolla, CA) in accordance with the description Marchuk and others (1991) Nucl. Acids Res. 19, 1154. Plasmid pBluescript (20 µg) was digested restricteduse the endonuclease EcoRV, and then purified by extraction with a mixture (50: 50, vol/about.) phenol and chloroform. After precipitation with ethanol, the DNA was treated with 9 units of Tag DNA polymerase for 2 hours at 70oC in 50 ál reaction containing 50 mm KCl, 10 mm Tris-HCl (pH 9,0), 0.1% Triton-X100, 1.5 mm MgCl2and 2 mm dTTP. Then the vector was again purified by extraction with phenol and chloroform (50: 50, vol/about.) and precipitated with ethanol. The result of this procedure was the addition of a single deoxythymidine balance to each 3'-end that allows the use of this vector for the insertion of DNA fragments synthesized by Tag DNA polymerase.

Nefosfaurilirovanna PCR fragment of 450 p. O. inserted into T-vector by reaction with DNA digati T4 (New England Biolabs, Beverly, MA), using conditions recommended by the manufacturer. Ligious the reaction mixture is incubated for approximately 72 hours at 16oC, and then the Wali on LB-agar, containing 50 μg/ml ampicilin. Before seeding the cells on the surface of the agar of each Cup was applied by spraying 100 μl of 2% X-gal (Life Technologies, Inc., Grand Island, NY) and 40 μl of 100 mm IPTG (Life Technologies, Inc., Grand Island, NY) and left for drying. After incubation overnight at 37oC were visible colonies, painted blue pigment, and uncolored colonies (white). From cultures with 12 white colonies were isolated and plasmid DNA. All 12 isolates contained the insert 450 p. O. For further analysis were selected 5 clones: 3CB4, 3CB6, 3CB7, 3CB8 and 3CB9. Analysis of DNA sequences was performed using sequenase set "Sequenase version 2.0 of" (United States Biochemical, Cleveland, Ohio).

Procedure 5'-RACE clone

5'-RACE system for rapid amplification of cDNA ends was purchased from Life Technologies, Inc. , Grand Island, NY. Poly+RNA uterus person purchased from Clontech Laboratories, Palo alto, CA. Experiments on the 5'RACE cloning was performed using the Protocol and reagents supplied with 5'-RACE system, except that: (a) CKCB7-, ACP and UAP-primers were synthesized on an oligonucleotide synthesizer model 392, Applied Biosystem. Inc. and used as described for 3'-RACE clone; and (b) thermal Cycling PCR program "Touchdown" described for 3'-RACE clone used for DVS is from 0.5 ál of 10 mm solution CKCB7 and 13.5 μl of DEPC-treated water, and the resulting mixture was heated at 70oC for 10 minutes. After cooling the mixture on ice was added to the reaction components so that the final composition contained approximately: 20 nm Tris-HCl (pH 8,4) 50 mm KCl, 3 mm MgCl2, 10 mm DTT, 200 mm CKCB7, 400 μm each of dATP, dCTP, dGTP and dTTP, and 40 ng/μl of RNA in a volume of 24 μl. The reaction mixture was heated to 42oC, was added 1 μl (220 units) reverse transcriptase II. After a 30 minute incubation at 42oC and a 15-minute incubation at 70oC the reaction mixture was placed in conditions 55oC and added 1 μl (2 units) RNase H. Digestion by RNase H was carried out for 10 minutes at a temperature of 55oC.

cDNA was separated from uninvolved dNTPS, CKCB7 and proteins using the Glassmax DNA Isolation Spin Cartridge (included in 5'-RACE system). In particular, 120 μl of a solution for binding (6 M NaI) was added to the reaction mixture of the first thread, and cDNA/NaI solution was transferred into a centrifuge tube GIASS MAX. After centrifugation at 13,000 g for 20 s was added 0.4 ml of cold (4oC) wash buffer. Then centrifugally still 20 at 13,000 g. This step was repeated two more times. After twice washing with 400 μl of cold (4oC) 70% ethanol cDNA was suirable by adding centrifuge PR the polymer tail added to the 3'-end of the cDNA using terminal deoxynucleotidyltransferase (TdT) and dCTP. This reaction was carried out in a PCR-compatible buffer. 10 μl of purified cDNA was combined with 7.5 ál of DEPC-treated water, and 2.5 μl of 10 x reaction buffer and 1.5 μl of 25 mm solution of MgCl2and 2.5 µl of 2 mm dCTP solution. The reaction mixture is incubated for 2 to 3 minutes at 94oC. After cooling for 1 minute on ice was added 1 μl TdT (10 units /ál). The final mixture had the following composition: 10 ál of cDNA in 20 mm Tris-HCl (pH of 8.4), 50 mm KCl, 1.5 mm MgCl2, 200 μm dCTP, and 0.4 units/μl TdT. The reaction mixture was incubated for 10 minutes at 37oC and then for 10 minutes at 70oC for inactivation of TdT.

For the implementation of PCR were prepared with four different reaction mixtures having the following final concentration of primer (50 ál):

1. 400 nm ACP

2. 800 nm ACP

3. 360 nm UAP and 40 nm ACP (UAP:ACP = 9:1)

4. 720 nm UAP and 80 nm ACP (UAP:ACP = 9:1)

Final concentration (50 µl) of the other components in all four reactions were identical: 5 ál of poly-A+oC-terminal cDNA in 20 mm Tris-HCl (pH 8,4); 50 mm KCl 1.5 mm MoCl2: 400 nm CKCB2, and 200 μm each of dATP, dCTP, dGTP and dTTP. Components, including ACP and UAP-primers were mixed in the original volume of 45 μl and heated to 94oC thermoacetica. Then to each reaction was added 5 μl of a mixture of 1.25 units of DNA polanc fragment used the PCR thermal Cycling "Touchdown".

After PCR-amplification of all four reaction mixtures were combined and the DNA products were fractionally by size by electrophoresis on 8% polyacrylamide gel. The DNA product of about 230 p. O., cut out from the gel, purified and subcloned into the T-vector, as described above for the 3'RACE fragment 450 p. O. For further analysis identified 5 clones: 5CB2, 5CB3, 5CB5, 5CB6, 5CB11. Analysis of DNA sequences was performed using sequenase set "Sequenase version 2.0" (United States Biochemical, Cleveland, Ohio).

In Fig. 8 shows the complete cDNA sequence of the Component B, collected from a RACE of clones 5CB3 and 3CB7, which contains restriction sites.

The analysis of the primary structures of the cDNA sequence of clone 5CB3, 5CB6, 5CB11 and 3CB4, 3CB7, 3CB9 have shown full compliance with these sequences, the exon Component B (genomic clone 4D Example 3).

Although the present invention is described above with specific examples of its implementation, however, experts in this field it is obvious that it can be made various changes or modifications not beyond being and scope of the following claims.

Labels to drawings

Fig. 1. The working scheme for the purification of Component B from mponent B shown in SEQ ID No. 2). The arrows show the sites of splicing.

Fig. 3. The sequence of the promoter region of the Component B (the promoter region of the specified Component B shown in SEQ ID No. 2), the binding sites for the transcription factors AP-1, AP-2, Sp-1 and E-blocks are specified. It is also shown TATA-block and GPE.

Fig. 4. Restriction map of the gene of Component B. Derived mRNA shows a line below genomic gene: the area marked in the frame, denote a sequence encoding a protein.

Fig. 5. Restriction map of the insert of clone 4D.

Fig. 6. Restriction map of the plasmid pBSCB4D.

Fig. 7. The General scheme used for RACE-cloning the DNA sequence of Component B.

Fig. 8. The complete cDNA sequence of the Component B, which shows the restriction sites (cDNA specified Component B is represented in SEQ ID No. 3).

1. The polypeptide inhibits the binding of TGF-alpha to its receptor, characterized by the amino acid sequence of SEQ ID No : 1, or its salt of carboxylic functional groups and amine groups having the same biological activity.

2. Drug having anti-inflammatory and/or antikoaguliruyuschey, and/or antitumor and what I polypeptide under item 1, characterized in that conduct: (a) adsorption of urine at acid pH on the kaolin and its extraction with ammonia; b) elution of fraction (a), resin Bio-Rex 70 using ammonia; (C) elution of fraction (b) on DEAE-Sepharose resin using acetate buffer; (d) elution of fraction (C) for CM-Sepharose resin using acetate buffer; (e) elution of fraction (d) in the resin LC 18 (HPLC) using a mixture of acetate buffer and acetonitrile; (f) the elution fraction (e) in the resin DE-52 using acetate buffer; (g) elution of fraction (f) for resin D-Zephyr using acetate buffer; (h) elution of fraction (g) on the resin 18 HPLC (HPLC) using a mixture of water triperoxonane acid and acetonitrile; (i) elution of fraction (h) resin D-Zephyr using acetate buffer.

4. The method according to p. 3, characterized in that specified in the urine is the urine of man.

5. The DNA fragment encoding the polypeptide under item 1.

6. The DNA fragment encoding the polypeptide under item 1 and capable of gibridizatsiya with the DNA fragment under item 5.

7. A fragment of genomic DNA encoding the polypeptide under item 1, characterized by the nucleotide sequence of SEQ ID No:2.

8. Fragment to DNA encoding vector, including the DNA fragment under item 5.

10. Expressing a vector comprising the DNA fragment under item 6.

11. Expressing a vector comprising the DNA fragment under item 7.

12. Expressing a vector comprising the DNA fragment under item 8.

13. The strain of E. coli, transformed expressing vector under item 9 and producing the polypeptide under item 1.

14. The strain of E. coli, transformed expressing vector at p. 10 and producing the polypeptide under item 1.

15. The strain of E. coli, transformed expressing vector for p. 11 and producing the polypeptide under item 1.

16. The strain of E. coli, transformed expressing vector for p. 12 and producing the polypeptide under item 1.

17. The method of producing the polypeptide under item 1, comprising culturing prokaryotic or eukaryotic cells transformed by expressing the vector according to any one of paragraphs.9-12, and the secretion of the polypeptide from the culture medium.

18. Basically pure protein containing the polypeptide under item 1 or its salts carboxylic groups or functional amine groups having the same biological activity, obtained by the process comprising the following stages: a) adsorption of urine at acid pH on cholinergie (b) on DEAE-sepharose resin using acetate buffer; (d) elution of fraction (C) for CM-Sepharose resin using acetate buffer; (e) elution of fraction (d) in the resin LC 18 (HPLC) using a mixture of acetate buffer and acetonitrile; (f) elution fraction (e) in the resin DE-52 using acetate buffer; (g) elution of fraction (f) for resin D-Zephyr using acetate buffer; (h) elution of fraction (g) on the resin 18 HPLC (HPLC) using a mixture of water triperoxonane acid and acetonitrile; (i) elution fractions (h) resin D-Zephyr using acetate buffer.

19. Pharmaceutical composition having a property to inhibit the binding of TGF-alpha (growth factor of T cells), characterized in that it contains a therapeutically acceptable amount of a polypeptide under item 1 in combination with one or more therapeutically acceptable excipients or diluents.

20. Pharmaceutical composition having a property to inhibit the binding of TGF-alpha (growth factor of T cells), characterized in that it contains a therapeutically acceptable amount of basically pure protein by p. 18 in combination with one or more therapeutically acceptable excipients or diluents.

 

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