Method of producing recombinant spider-web protein, fused protein, recombinant dna, expression vector, host cell and producer strain

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing recombinant spider-web proteins of orb-weaving spiders in yeast cells, fused proteins containing spider-web recombinant protein sequences of orb-weaving spiders, recombinant DNA encoding fused proteins, yeast host cells and expression vectors used to realise the method, as well as producer strains of recombinant proteins of orb-weaving spiders. The method involves constructing an expression vector containing a DNA sequence encoding recombinant spider-web proteins of orb-weaving proteins, fused with a sequence which encodes ubiquitin or an ubiquitin-lie protein SUMO of yeast Saccharomyces cerevisiae, which occupies the N-terminal position in the fused protein relative the recombinant spider-web protein, transformation of yeast cells with the obtained expression vector and expression of orb-weaving spider web protein in the transformed cells.

EFFECT: method increases production of recombinant spider-web protein during accumulation thereof in yeast cells in a water-soluble fraction in form of a protein which does not contain a hybrid component.

24 cl, 6 dwg, 12 ex

 

The technical field to which the invention relates.

The invention relates to the field of biotechnology and is directed to a method of obtaining a recombinant protein of the webs of Orb-web spiders, fused proteins comprising sequences of the recombinant proteins of the webs of Orb-web spiders and sequence of the ubiquitin-like proteins, recombinant DNA encoding the fused protein, the host cell of yeast expression vectors used for implementing the method, and strains-producers of recombinant proteins webs of Orb-web spiders.

The level of technology

The web is a unique biomaterial that combines amazing strength and elasticity. For these indicators it has no analogues in nature, and among the materials created by man. So, for example, frame thread of a web spider crporate Nephila clavipes by the values of tensile strength superior to steel and comparable to Kevlar, and the largest energy gap exceeds and Kevlar; at the same time, it may take up to 35% of its length [Gosline J.M. et al. Endeavor, 1986, v.10, 37-43].

Getting industrial quantities of such materials is possible only with the help of genetic engineering and biotechnological methods. To date, several genes encoding proteins of the web, dedicated and adequately characterized [Xu M & Lewis R. Proc. Natl. Acad Sci., USA, 1990, v.87, 7120-7124; Hinnman M. & Lewis R. J. Biol. Chem., 1992, v.267, 19320-19324; Guerette, P. et al. J. Science 1996, v.272, 112-115; Hayashi, C.Y. & Lewis R.V. J. Mol. Biol., 1998, v.275, 773-784]. These genes belong to the most extensive known Castronovo (sizes mRNA lie in the range from 7.5 to 15.5 called) and consist of a large number of tandemly repeated long sequences that differ markedly different genes. The most studied frame thread of spider-crporate Nephila clavipes consists of two proteins - spidroin 1 and spidroin 2 (MaSp1 and MaSp2, respectively)synthesized a large inuloides gland [Hinnman M. & Lewis R. J. Biol. Chem., 1992, v.267, 19320-19324; Guerette, P. et al. Science, 1996, v.272, 112-115]. A recurring element spidroin 1 can be represented as the following consensus sequence:

[GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)4-7],

and a repeating sequence of spidroin 2 -

[GPGGYGPGQQGPGGYAPGQQPSGPGS(A)6-10],

The principal difference between these proteins is that if spidroin 1 elementary repetition is the Tripeptide GGX (X=A, S or Y), as in the case of spidroin 2 - Pentapeptide GPGGX and GPGQQ. For spidroin 1 characterized by increased strength, and to spidroin 2, capable of forming a β-spiral [Hayashi et al., 1999, Int. J. Biol. Macromol., v.24, 271-275], - greater flexibility. The interaction of these proteins frame thread web and provides a unique combination of properties.

Bel and MiSp1 and MiSp2, synthesized low inuloides gland, and protein Flag hunting thread of spider-crporate also have a repetitive structure. Repeating regions enriched in alanine and glycine. The GGX motifs and GA are presented for the entire length of the amino acid sequence as the protein MiSp1, and protein MiSp2 [K.Vasanthavada et al. Cell. Mol. Life Sci, 2006, v.63, 1986-1999]. In protein sequence Flag hunting thread dominant recurring motifs represented by Pentapeptide GPGGlX and Tripeptide GGX.

The results of the study of proteins frame thread of spider-crporate Nephila clavipes, and squirrel hunting thread and proteins, synthesized low inuloides gland [Kohler T. & Vollrath F. J. Exp. Zool., 1995, v.271, 1-17; Colgin M.A. & Lewis R., Protein Sci., 1998, v.7, 667-672], allowed us to propose a modular hypothesis of the structure of proteins web [Hinman at al., 2000, TIBTECH, v.1, 374-379]. Structural analysis of proteins-wide-web indicates the presence of crystalline regions formed by β-folded structures (it is believed that they are formed by the blocks (A)nand (GA)n), which provide strength to the fibers in the web and are shipped in less structured Gly-rich matrix, responsible for elasticity. At the ends of the molecules are non-recurring (NR) unique conserved sequence that is necessary is believed to increase the solubility of proteins in concentrated solution inside the drive, and for the correct fitting of the molecules during the formation of filaments during the spinning.

Attempts were made to clone and optimization of expression of cDNA copies of natural protein-coding genes, frame thread web, in E. coli cells [S. Arcidiacono et al. Appl. Environ. Biotechnol., 1998, v.49, 1-38]. However, the achieved level of expression was quite low, which is explained, first of all, the discrepancy in the frequency of occurrence of certain amino acid codons in the genes of a spider and the genes used microorganism recipient.

More successful was the way the chemical-enzymatic synthesis of the protein coding genes of the web with subsequent cloning in bacteria, yeast, tobacco, potatoes, using a synthetic DNA modules with frequency of use of codons, adapted to the respective host cell [Prince J.T. et al. Biochemistry, 1995, v.34, 10879-10884; S. Winkler et al. Int. J. Biol. Macromol, 1999, v.24, 265-270; Fahnestock S.R. & Bedzyk L.A. Appl. Environ. Biotechnol, 1997, v.47, 33-39; Fahnestock S.R. & Irwin S.L., Appl. Environ. Biotechnol., 1997, v.47, 23-32; Lewis, R.V. et al., 1996, Protein Expr. Purif., v.7, 400-406; Scheller, J. et al., 2001, Nat. Biotechnol., v.19, 573-577]. These works were mainly concerned with the expression of genes encoding recombinant proteins frame threads of webs of Orb-web spiders, containing the consensus sequence or small fragments. As a result of expression of the synthetic genes were obtained artificial proteins that contain Amie options primary retry spidroins 1 and 2, similar to the recurring areas of natural proteins. These proteins although it possessed the characteristics of secondary structure, characteristic of the web, but created on the basis of the threads on the mechanical properties were far from natural fibres of the web. None of these artificial proteins did not contain C-terminal NR-regions, which are found in all proteins frame threads. Properties closest to the properties of natural proteins, possessed artificial analogues containing 800 and 1600 amino acid residues, resulting from the expression of synthetic genes in E. coli cells [Fahnestock & Irwin, 1997, Appl. Environ. Biotechnol., v.47, 23-32] or yeast Pichia pastoris [Fahnestock & Bedzyk, 1997, Appl. Environ. Biotechnol, v.47, 33-39].

The first step in the direction of the studies of molecular mechanisms of Assembly of web threads was to study the primary structure of the skeleton proteins ADF-3 and ADF-4 wide web of the garden spider (Araneus diadematus), corresponding MaSp2 proteins MaSp1 and (spidroin 2 and 1 large inuloides gland). Recombinant proteins web composed of synthetic repetitive sequences and unique authentic NR-regions at the ends of the molecules were expressed in cells of E. coli, the yield of purified protein was approximately 1 g per liter of bacterial culture [WO/2006/008163].

A comparative analysis of secondary structure, solubility and aggregation properties of the obtained be the Cove has allowed to identify the role of different elements of the primary structure of proteins the web. It was found that the repeating region, occupying the major length portion of the web and proteins containing the consensus sequence, including poly-a block, determine the solubility of synthetic proteins. Important to ensure solubility is the alternation of hydrophobic and hydrophilic segments in the primary repetitions.

Non-repeating region From the end of the play an important role in initiating the Assembly of proteins. In this expression system were used as synthetic building blocks of two modules: module poly-a and the second module, consisting of four repeats GPGQQ. The modules of this type are also described Hammerich with co-authors [Hummerich, D. et al., 2004, Biochemistry, v.43, 13604-13612].

The Assembly process of the web has been studied in models of two recombinant analogues of spidroin 1 (protein 1F9) and spidroin 2 (protein A), members of the frame threads of the web of the spider Nephila clavipes and Nephila madagascariensis, respectively [Bogush V.G. & Debabov V.G., 2009, J. Neuroimmune Pharmacol., v.4, 17-27].

Synthetic gene protein 1F9 was expressed in the yeast Saccharomyces cerevisiae under the control of the GAL1 promoter using byreplacing expression vector [Bogush V.G. et al., 2001, Biotechnology, Vol.2, p.11-22] and in cells of methylotrophic yeast Pichia pastoris under the control of methanosarcinales AOXI promoter using the integrative vector pHIL-D2 [Bogush V.G. et al., 006, Biotechnology, 4, 3-12].

In the first case, more than 80% of the target protein was detected in the insoluble fraction, and the average yield amounted to 6-8 mg of protein per 1 liter of fermentation yeast culture. In the yeast Pichia pastoris average yield of pure protein 1F9 was approximately 70 mg per 1 kg of wet cell mass (approximately 23 mg/l fermentation culture). The sequence of the recombinant proteins were similar to sequences of natural proteins, in particular, the repeating region of the protein 1F9 contained 9 repetitions of "monomer", consisting of five variations of the primary repetitions found in natural spidroin 1. To increase the level of synthesis of the recombinant protein in yeast cells the structure of genes 1f9 and E was modified by replacing the "rare" triplets on codons characteristic effectively expressed genes of yeast, and the number of internal repeats of nucleotide sequences is minimized. DNA fragments encoding the corresponding monomers of both proteins were obtained by chemical-enzymatic synthesis and then amplified. The final gene protein 1F9 encode nine repetitions of the corresponding "monomer"that make up a protein with a molecular mass of 94 kDa; protein A (molecular mass of 113 kDa) contained 12 "Monomeric" replays.

In solutions 1F9 and E, purified using cation-exchange chromatography were investigated structural transitions that occur when certain actions [Bogush V.G. & Debabov V.G., 2009, J. Neuroimmune Pharmacol., v.4, 17-27]. Despite the absence of a hydrophilic N - and C-terminal unique sequences (NR), which, as previously thought necessary for the formation of nanofibrils and micelles, both proteins in aqueous solution spontaneously formed nanofibril length of 100 nm - 1 μm and micelles with a diameter of about 1 μm. And nanofibrillar had a spiral structure with a period of 40 nm.

However, the level of synthesis of recombinant proteins the web using known methods did not allow to produce proteins of the web in quantities sufficient not only to study their structure and properties, but also for the development and testing of a new class of medical materials and products from them. One way to increase expression of poorly expressed protein by genetic engineering methods is its biosynthesis in the form of a hybrid protein in which the target protein is fused with effectively expressed protein [Shatzman and Rosenberg, 1987, Methods Enzynol., v.152, 661-673]. Typically, however, the advantages of this approach largely offset by the need in the final stages of cleaning to carry out the processing of the hybrid product for release from his membership of the target protein, h is about ethnologica in the case of recombinant proteins the web. It was shown that when using yeast ubiquitin as effectively expressed component hybrids are highly specific intracellular processing in yeast cells. Ubiquitin yeast, consisting of 76 amino acid residues, is a representative of the family of ubiquitin-like proteins in eukaryotes, which includes a relatively small structurally conservative proteins with extraordinary speed folding, high solubility and heat stability; in vivo proteins of this family are used for reversible modifications and changes in the functional status of other proteins. Members of the family of ubiquitin-like proteins contain conservative C-terminal motif Gly-Gly, which is the site of processing [Müller et al., 2001, Nature, v.2, 202-210]. The presence of this site in the hybrid proteins with ubiquitin leads to the fact that in yeast cells hybrids are highly specific intracellular processing under the action of ubiquitin-specific proteases DUB, resulting in the structure of final products expression ubiquitinates component is not available.

It was later discovered that the addition of ubiquitin to increase expression can be used, and other representatives of proteins ubiquitinated family, in particular, yeast variant protein SUMO. Mature the SUMO protein of the yeast Saccharomyces cerevisiae, encoded by a unique gene SMT3 (Johnson et al., 1997, EMBO J, v.16, 5509-5519; Muller et al., 1998, EMBO J, v.17, 61-70), contains in its composition of 98 amino acid residues, of which the remains 13-98 important for the formation of the native structure [Mossessova E. & Lima CD., 2000, Mol. Cell, v.5, 865-876]. Similar to ubiquitin-specific proteases, SUMO-specific proteases yeast provides high efficiency and specificity processing SUMO-containing hybrid proteins [Malakhov et al., 2004, J. Struct. Funct. Million., v.5, 75-86; Butt et al., 2005, Protein Expr. Purif., v.43, 1-9].

However, the efficiency of hybridization of recombinant proteins the web with ubiquitin and other ubiquitin-like protein to increase protein biosynthesis wide web has not been demonstrated. Thus, development of a method of microbial biosynthesis, which greatly improve the production of recombinant proteins on the web, with properties close to the properties of natural proteins is an important task which opens in principle possible to create on the basis of recombinant proteins web biomaterials with unique properties.

Disclosure of inventions

The authors of the present invention for the first time a method for obtaining recombinant proteins webs of Orb-web spiders (Araneidae) in yeast cells, providing the production of recombinant proteins in Koli is the EU ETS, in tens times more than the number of recombinant proteins webs produced in accordance with methods known from the prior art.

According to the proposed method of recombinant proteins webs of Orb-web spiders Express in yeast cells in the form of a hybrid with the ubiquitin-like protein, which occupies in the structure of the hybrid N-terminal position and contains the site of processing, recognized natural yeast proteases, preferably, the ubiquitin-specific proteases DUB or SUMO-specific proteases yeast, resulting in the expression of hybrid proteins are subjected to processing under the action of proteases, which provides accumulation in yeast cells of the Mature protein of the web, not containing hybrid component, and the protein accumulates in the water-insoluble fraction of yeast cells.

Preferably, the method according to the invention provides for recombinant protein wide web, the consensus sequence of which come from proteins frame large inuloides cancer and/or small proteins inuloides cancer, or squirrel hunting thread of spider-crporate.

In one of preferred embodiments of the method according to the invention provides for recombinant proteins wide web, the consensus sequence of which PR is coming from proteins frame large inuloides gland of Nephila clavipes and/or Nephila madagascariensis, and the ubiquitin-like protein selected from the group including ubiquitin and SUMO protein of the yeast Saccharomyces cerevisiae.

In one of the most preferred embodiments of the method according to the invention aims to obtain recombinant protein A frame thread of spider-crporate Nephila madagascariensis in cells of Saccharomyces cerevisiae under the control of the promoter of the gene GAL1 yeast, and gene recombinant protein fused with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In another most preferred embodiment of the method according to the invention is directed to gene expression recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes in cells of Saccharomyces cerevisiae under the control of the promoter of the gene GAL1 yeast, and gene recombinant protein fused with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In one aspect the invention is directed to a protein comprising the sequence of the recombinant protein the web of spider-crporate and ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and the sequence of the recombinant protein of the web includes a consensus sequence that occur from repeated sequences of proteins of frame threads MaSp1 and MaSp2 great inuloides gland, b is lcov MiSp1 and MiSp2 small inuloides cancer and protein Flag hunting thread of spider-crporate.

In one of the preferred embodiments the invention is directed to a protein, in which the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein of the web comprises a consensus sequence derived from repetitive sequences of proteins of frame threads MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

Most preferably, the protein includes a recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes, the sequence of which is fused with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In another most preferred embodiment of the protein includes a recombinant protein A frame thread of spider-crporate Nephila madagascariensis, the sequence of which is fused with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In one aspect the invention is directed to recombinant DNA encoding a protein comprising the recombinant protein of web of spider-crporate and ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and the sequence of the recombinant protein web VK is uchet consensus sequences, which are derived from repetitive sequences of proteins of frame threads MaSp1 and MaSp2 great inuloides gland, protein MiSp1 and MiSp2 small inuloides cancer and squirrel hunting thread of spider-crporate.

Preferably, the recombinant DNA encodes a protein in which the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein the web of spider-crporate includes a consensus sequence derived from repetitive sequences of proteins MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

Most preferably, the recombinant DNA according to the invention encodes a protein including the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes and ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and having the sequence as shown in the sequence Listing (SEQ ID NO: 1 and SEQ ID NO: 3, respectively).

In another most preferred embodiment of the recombinant DNA encodes a protein including the sequence of the recombinant protein A frame thread of spider-crporate Nephila madagascariensis and ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In one aspect of the invention relates to vectors ex is ressie, which include DNA sequences encoding recombinant proteins the web of spider-crporate, merged with the sequence of the gene ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and sequence highly regulated yeast promoters.

In the preferred embodiment of the invention provides expression vectors that contain recombinant DNA encoding a protein in which the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein of the web comprises a consensus sequence derived from repetitive sequences of proteins MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

In one of the most preferred embodiments of the invention provides an expression vector that represents perepilichny vector pPDX3-HUB-2E12, containing an area of replication initiation endogenous 2 μm plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein A, the merged sequence that encodes a ubiquitin Saccharomyces cerevisiae.

In another most preferred embodiment izobreteyonija the expression vector, representing perepilichny vector pPDX3-HUB-1F9, containing an area of replication initiation endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein 1F9, merged with a sequence that encodes a ubiquitin Saccharomyces cerevisiae.

In another most preferred embodiment of the invention provides an expression vector that represents perepilichny vector pPDX3-SUMO-1F9, including scope for replication initiation of endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein 1F9, merged with a sequence that encodes a protein SUMO Saccharomyces cerevisiae.

In accordance with another aspect of the invention provides host yeast cells producing recombinant proteins the web of spider-crporate. The most preferred host cells according to the invention are cells of Saccharomyces cerevisisae. In another aspect the invention provides strains-producers of recombinante protein 1F9 and A frame thread of spider-crporate.

Brief description of drawings Fig 1. Electrophoresis in 12% SDS page with DDS-Na fractions 1F9 after chromatography on a cation exchange column (HiPrep 16/10 SP FF. Tracks: 1 - original solution before application to the column; 2 - breakthrough; 3-6 - fractions containing protein 1F9, 7 - example of the conventional 1F9. Figure 2. Electrophoresis in 12% SDS page with DDS-Na fractions E after chromatography on a cation exchange column (HiPrep 16/10 SP FF. Tracks: 1 - Original solution before application to the column; 2 - skip; 3 - Standards of molecular weights (from top to bottom, in kDa): 170, 130, 95,72, 55, 43, 34, 26, 17; 4 - the fraction containing E, 5 - sample standard E. Figure 3. Map vector pPDX3-HUB-1F9.

Legend: SPIDROIN - synthetic gene of the recombinant protein 1F9 (spidroin-1 spider N. clavipes); HUB - gene ubiquitin yeast S.cerevisiae; GAL1 - promoter region of the gene GAL1 yeast S.cerevisiae; URA3 and PGK1 - structural genes URA3 and PGK1 yeast S.cerevisiae, respectively; cyc1T sequence of the transcription terminator CYC1 gene of the yeast S.cerevisiae; 2 mkm - a fragment of the endogenous 2 μm plasmid of the yeast S.cerevisiae, containing the starting area replication; pUC18 - fragment of plasmid pUC18 containing the gene for beta-lactamase (ApR) and the area started replication, ensuring selective amplification of the vector in cells E.coli.

Figure 4. Map vector pPDX3-SUMO-1F9.

Legend: SPIDROIN - synthetic gene of the recombinant protein 1F9 (recombinant spidroin-1 spider N. clavipes); SUMO - SMT3 gene of the yeast S.cerevisiae, encoding protein SUMO; GAL1 - promoter region of the gene GAL1 yeast S.cerevisiae; URA3 and PGK1 - structural genes URA3 and PGK1 yeast S.cerevisiae, respectively; cyc1T sequence of the transcription terminator CYC1 gene of the yeast S.cerevisiae; 2 mkm - a fragment of the endogenous 2 micron the plasmid of the yeast S.cerevisiae, contains the starting area replication of yeast; pUC18 - fragment of plasmid pUC18 containing the gene for beta-lactamase (ApR) and the starting area replication to provide selective amplification of the vector in cells of E. coli. Figure 5. Scheme vector pPDX3-HUB-2E12.

Legend: SPIDROIN - DNA sequence encoding a recombinant protein A; HUB - a DNA sequence encoding a ubiquitin yeast S.cerevisiae; GAL1 - promoter region of the gene GAL1 yeast S.cerevisiae; URA3 and PGK1 - structural genes URA3 and PGK1 yeast S.cerevisiae, respectively; cyc1T sequence of the transcription terminator CYC1 gene of the yeast S.cerevisiae; 2 mkm - a fragment of the endogenous 2 μm plasmid of the yeast S.cerevisiae, containing the starting area replication; pUC18 - fragment of plasmid pUC18 containing the gene for beta-lactamase (ApR) and the area started replication, ensuring selective amplification of the vector in E. coli cells. 6. Photo of artificial filaments of protein 1F9 in a vessel with ethanol.

The implementation of the invention

The present invention is based on the surprising discovery that the expression of the recombinant protein the web of spider-crporate in yeast cells in the form of a fused protein with ubiquitin-like protein, which occupies in the structure of the hybrid N-terminal position, allows dozens of times to increase the production of recombinant protein a web, and a recombinant protein, expressed in the form of hibri the nogo protein, accumulates in the cells of the yeast in the water-insoluble fraction in the form processioning protein that does not contain a hybrid component.

Therefore, in one aspect the present invention provides a method of obtaining a recombinant protein of web of spider-crporate in yeast cells, providing for the construction of the expression vector, transformation of yeast cells obtained expression vector and expression in transformed cells gene recombinant protein of web of spider-crporate, characterized in that use expression vector which includes a DNA sequence encoding a recombinant protein of web of spider-crporate, merged with a sequence that encodes a ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and contains the site of processing, recognized natural yeast proteases preferably, the ubiquitin-specific proteases DUB or SUMO-specific proteases yeast, resulting in the expression of hybrid proteins are subjected to processing under the action of proteases, which provides accumulation in yeast cells in water-insoluble fraction of the recombinant protein of the web in the form of processioning protein that does not contain a hybrid component.

Recombine the major proteins, obtained by the method according to the invention have a marked periodicheskoy structure that can be represented in the form of a number of consensus sequences inferred by the alignment of the repeating units of natural proteins webs of Orb-web spiders. Recombinant proteins according to the invention are proteins, sequences which contain occurrences of a consensus sequence and combination of repeats of the consensus sequences of various types, originating from proteins frame large inuloides cancer and/or small proteins inuloides cancer, and/or proteins Flag hunting thread of spider-crporate, in particular selected from the group comprising the consensus sequence:

where MaSp1 and MaSp1 - proteins frame strand of large inuloides cancer Latrodectus hesperus [Lawrence V.A. et al., 2004, Biomacromolecules, v.5, 689-695];

MiSp1 and MiSp1 - protein low inuloides gland of Nephila clavipes [Colgin M.A. & Lewis R.V., 1998, Protein Sci., v.7, 667-672];

Flag - squirrel hunting thread Nephila madagascariensis [Hayashi, C. & Lewis R.V., 1998, J. Mol. Biol., v.275, 773-784].

Preferably, according to the proposed method using consensus sequences derived from repetitive sequences of proteins of great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

Construction of synthetic genes encoding recombinant proteins is large and/or small ampulove glands, or proteins Flag hunting thread of spider-crporate, includes the reconstruction of the DNA sequence that encodes a consensus sequence or a combination of repeats of the consensus sequences of various types, originating from the repetitive sequence of the above protein; design and chemical synthesis of a series of primers to the consensus sequences/sequences; one-time annealing a mixture of all the synthesized primers necessary for the formation of double-stranded DNA molecules, and the subsequent processing of their ligase to remove single-stranded DNA breaks, or a PCR reaction with the consistent use of appropriate primers and phased completing the growing DNA fragment, moreover, the formed fragment ("monomer") then undergoes a gradual doubling in the structure of the plasmid to obtain a gene required length [Bogush V.G. et al., 2001, Biotechnology, Vol.2, 11-22; Bogush V.G. et al., 2006, Biotechnology, V. 4, 3-12; Bogush V.G. & Debabov V.G., 2009, J. Neuroimmune Pharmacol., v.4, 17-27].

The sequence of the corresponding cDNA can be derived from the sequence of the natural protein, taking into account the degeneracy of the code and the frequency of occurrence of codons in yeast. In particular, when the design is funding the gene encoding a protein 1F9 and contains 9 copies of "monomer", fragments encoding the most common primary repetitions were selected from the sequence of the natural protein and differed from each other by a set of deletions. The reconstructed DNA sequence consisted of approximately 400 BP and encodes a polypeptide corresponding to a 134 amino acid residues. "Rare" codons in the sequence synthetic gene were replaced by the most commonly used in yeast. "Monomer" was obtained using chemical-enzymatic synthesis and multimeric form obtained by step-by-step animation of the monomer in the composition of the recombinant plasmids [Bogush V.G. et al., 2001, Biotechnology, Vol.2, 11-22].

When designing gene E were used sequence spidroins type 2 large inuloides gland contained in the database of protein sequences in NCBI and includes more than 200 amino acid residues. On the basis of mathematical analysis all sequences were designed sequence of blocks (each consisted of 3-5 primary repetitions) and made up formula full synthetic gene [Bogush V.G. et al., 2009, J. Neuroimmune Pharmacol., v.4, 17-27].

In one of preferred embodiments of the proposed method of producing a recombinant protein of web of spider-crporate in yeast cells involves the influence of the gene of the recombinant protein of the web with the DNA sequence, encodes a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae.

In one of the most preferred embodiments of the invention in cells of Saccharomyces cerevisiae receive recombinant protein 1F9 frame thread web of spider-crporate Nephila clavipes, and the structural gene of the protein 1F9 merged with the DNA sequence that encodes a ubiquitin Saccharomyces cerevisiae. In another most preferred embodiment of the invention in cells of Saccharomyces cerevisiae receive recombinant protein A frame thread web of spider-crporate Nephila madagascariensis, and gene protein E merged with the DNA sequence that encodes a ubiquitin Saccharomyces cerevisiae.

In yet another preferred embodiment of the invention in Saccharomyces cerevisiae yeast cells Express a fused protein containing the sequence of the recombinant protein 1F9, and the sequence of the protein 1F9 merged with the sequence of the protein SUMO yeast Saccharomyces cerevisiae. Recombinant proteins obtained according to the proposed method, were isolated from the water-insoluble fraction of the master cells of Saccharomyces cerevisiae using chromatography on a cation exchange column (examples 9 and 11). Electrophoretic analysis of the fractions (Figure 1 and 2) showed that the recombinant protein 1F9 and E accumulate in the fraction of water-insoluble proteins of yeast cells (in water-soluble fraction of recombinant proteins are practically absent is) and do not contain a component of the ubiquitin-like protein. This is indicated by electrophoretic mobility of the analyzed proteins and the absence of gel bands corresponding to the higher mobility of fused proteins (ubiquitin-1F9 and ubiquitin-E). Similar results were obtained for recombinant proteins extracted and purified from the insoluble fraction of the master cells of Saccharomyces cerevisiae producing recombinant proteins wide web, merged with the SUMO protein. Production of recombinant proteins by cells of Saccharomyces cerevisiae is not less than 100 mg/l fermentation culture.

The absence of recombinant proteins produced in accordance with the proposed invention, the water-soluble fraction allows practically to avoid loss of protein during isolation and purification in contrast to the known method [Bogush V.G. et al., 2001, Biotechnology, Vol.2, 11-22], according to which only about 80% of the target protein was detected in the insoluble fraction.

Thus, when implementing the method of producing a recombinant protein of webs according to the invention a recombinant protein synthesized in the cells of Saccharomyces cerevisiae, is collected in the fraction of water-insoluble proteins in the form of processioning protein that does not contain a hybrid component, and cells expressing recombinant protein web, accumulate ten times more recombinant protein than in accordance with the methods of the local of the prior art.

Purified recombinant proteins of web of spider-crporate, according to the invention, capable of forming supramolecular structures of various types, in particular the analyzed proteins form is not soluble in water thread (Example 12, Fig).

In yet another aspect the invention is directed to fused proteins comprising sequences of the recombinant protein the web of spider-crporate and ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and the sequence of the recombinant protein of the web includes a consensus sequence that occur from repeated sequences of proteins MaSp1 and MaSp2 great inuloides gland, protein MiSp1 and MiSp2 small inuloides cancer and protein Flag hunting thread of spider-crporate.

In one of the preferred embodiments the invention is directed to a protein, in which the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein the web of spider-crporate includes a consensus sequence derived from repetitive sequences of proteins MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

Most of predpochtitel is but protein contains the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes, merged with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae, as shown in the sequence Listing (SEQ ID NO:1 and SEQ ID NO:3, respectively).

In another most preferred embodiment of the protein contains the sequence of the recombinant protein A frame thread of spider-crporate Nephila madagascariensis, merged with a sequence that encodes a ubiquitin or SUMO protein of Saccharomyces cerevisiae.

In yet another aspect, the invention encompasses recombinant DNA encoding the fused protein. Synthesized and cloned DNA sequences used in accordance with the invention for the preparation of recombinant proteins on the web, encode fused proteins comprising sequences of the recombinant protein the web of spider-crporate and ubiquitin-like protein, which is in the composition of the fused protein N-terminal position relative to the recombinant protein, and the sequence of the recombinant protein of the web includes a consensus sequence that occur from repeated sequences of proteins MaSp1 and MaSp2 great inuloides gland, protein MiSp1 and MiSp2 small inuloides cancer and squirrel hunting thread Flag spider crporate.

Preferably, recombinant the Naya DNA encodes a protein, in which the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein the web of spider-crporate includes a consensus sequence derived from repetitive sequences of proteins MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis and selected from the group:

Most preferably, the recombinant DNA used in accordance with the invention for the preparation of recombinant proteins on the web, encode recombinant protein 1f9 frame thread of spider-crporate Nephila clavipes or recombinant protein A frame thread of spider-crporate Nephila madagascariensis, the sequence of which is fused with a sequence that encodes a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, as shown in the sequence Listing (SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3). The DNA of the present invention also include coding sequences, taking into account the degeneracy of the code and the sequence used as primers in amplification reactions.

Proteins according to the invention receive in yeast cells using expression vectors, which include gene fused protein containing the sequence of the recombinant protein the web of spider-crporate and ubiquitin-like protein, and isoeffective regulated promoters of the yeast such promoters as GAL1, GPD1, CUP1. Suitable vectors for constructing expression vectors in accordance with the invention can be used epilimnia vectors containing the region of initiation of replication of endogenous 2-micron plasmid of yeast, which ensures their ability to be maintained in yeast cells in episomes mnogoseriynom condition.

In one of preferred embodiments of the invention construct expression vectors carrying recombinant DNA encoding a protein, where the ubiquitin-like protein is a ubiquitin or SUMO protein of the yeast Saccharomyces cerevisiae, and the sequence of the recombinant protein the web of spider-crporate includes a consensus sequence derived from repetitive sequences of proteins MaSp1 and MaSp2 great inuloides gland of Nephila clavipes and Nephila madagascariensis.

In one of the most preferred embodiments of the invention construct expression perepilichny vectors pPDX3-HUB-1F9 (Figure 3) and pPDX3-FIUB-2E12 (Figure 5), which are obtained by cloning the sequences of the structural genes of the protein or protein 1F9 E, respectively, plasmids pPDX3-FIUB, bearing in its composition under the control of the promotor region of yeast GAL1 gene of Saccharomyces cerevisiae ubiquitin, and in each case will be merged in one frame is read the follower of the spine of the structural gene of the protein or protein 1F9 E and gene ubiquitin.

Still one of the most preferred embodiments of the invention construct expression perepilichny vector pPDX3-SUMO-1F9, which is obtained by cloning the gene sequence of the protein 1F9 plasmid pPDX3-SUMO bearing in its composition under the control of the promotor region of GAL1 yeast structural gene SMT3 Saccharomyces cerevisiae that encodes a protein SUMO Saccharomyces cerevisiae, and the clone will be merged in one reading frame of the gene sequence of the protein 1F9 and SMT3 gene (Figure 4). The expression vectors pPDX3-HUB-1F9, pPDX3-HUB-2E12 and pPDX3-SUMO-1F9 contain the region of initiation of replication of endogenous 2-micron plasmid of yeast, which ensures their ability to be maintained in the cells of the yeast Saccharomyces. cerevisiae in episomes mnogoseriynom condition.

In accordance with one aspect of the invention provides host yeast cells producing recombinant proteins webs of Orb-web spiders. As suitable host cells for producing recombinant proteins, web use yeast cells, which are selected from the group including Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris and Schizosaccharomyces pombe. Preferred host cells are cells of Saccharomyces cerevisisae. Most preferably, as the host cells used recipient strain Saccharomyces cerevisiae D702, which is diploid, that is in charge of the AET increased stability of its expression characteristics. Saccharomyces cerevisiae D702 contains a homozygous mutation in the chromosomal alleles of the structural gene PGK1, the coding phosphoglycerate kinase, which ensures stable maintenance of the vector in environments containing any single carbon source assimilable by yeast Saccharomyces cerevisiae, and GAL80 gene encoding protein-repressor promoter GAL1, and homozygous mutation, leading to changes in the regulation of GAL4 gene encoding a protein activator of the GAL1 promoter, resulting is galaktotrophousa expression of genes under the control of the GAL1 promoter.

In one of the most preferred embodiments of the invention the cells of the recipient strain Saccharomyces cerevisiae D702 transformyour expression vector pPDX3-HUB-1F9. The resulting strain SCR-702-1F9 producing recombinant protein 1F9 frame thread web of spider-crporate Nephila clavipes deposited in Russian national Collection of Industrial Microorganisms (VKPM) as Saccharomyces cerevisiae strain VKPM Y-3583.

One of the most preferred embodiments of the invention the cells of the recipient strain Saccharomyces cerevisiae D702 transform expression vector pPDX3-HUB-2E12. The resulting strain SCR-702-2E12 producing recombinant protein A frame thread web of spider-crporate Nephila madagascariensis deposited in Russian national Collection of Industrial Micro the of organisms (PMBC) as Saccharomyces cerevisiae strain VKPM Y-3584.

One of the most preferred embodiments of the invention the cells of the recipient strain Saccharomyces cerevisiae D702 transform expression vector pPDX3-SUMO-1F9.

Characterization of strains-producers. Genotype:

SCR-702-1f9 (a/α lu2/lu2 IgA/ura3 trp1/trp1 gal80::LEU2/gal80::LEU2 lys7/LYS7 his3/HIS3 his4/HIS4 pgk1:URA3/pgk1:URA3 GAL4:(STA2p-GAL4,TRP1)/GAL4:(STA2p-GAL4, TRP1) STA2/STA2 suc°/SUC2)/pPDX3-1f9

SCR-702-2E12 (a/α leu2/leu2 ura3/ura3 trp1/trp1 gal80:LEU2/gal80:LEU2 lys7/LYS7 his3/HIS3 his4/HIS4 pgk1:URA3/pgk1:URA3 GAL4:(STA2p-GAL4, TRP1)/GAL4:(STA2p-GAL4, TRP1) STA2/STA2 suc°/SUC2)/pPDX3-2E12

Morphological features:

Under cultivation at 28°C for 48 hours on agar YPD medium of the following composition (in wt.%): peptone - 2, yeast extract - 1, glucose - 2, agar - 2, water - the rest, cells of strains-producers of Saccharomyces cerevisiae have an oval shape, 3-7 µm in diameter. Cells packouts. Budding true, multilateral. True mycelium is not formed. Colonies are the following:

1) on agar medium YPD colonies are white with a smooth edge and a matte surface, a lenticular profile and creamy consistency;

2) on agar medium with starch (composition in wt.%: peptone - 2, yeast extract - 1, starch - 1, agar - 2, water - the rest of the colony is white with a patterned edge, matte surface, lenticular profile and krupchato consistency.

Growth in liquid medium with starch: at 28°C during the first 24 h cultive the Finance - the liquid is cloudy, white precipitate, not crumpled, parietal film does not form.

Physico-chemical characteristics:

Both strains of facultative anaerobes. Temperature growth 20-33°C (optimum - 28°C). the pH of the cultivation of 3.8 to 7.4 (optimum - 5,0).

Assimilation of carbon sources:

Both strains fermented glucose, fructose, maltose, sucrose, dextrin, starch. Not fermented lactose, galactose, inulin, xylose, arabinose.

Assimilation of nitrogen sources:

Both strains metabolize amino acids, ammonium sulphate, ammonium nitrate.

Storage:

Strains were stored at -70°C in 20% aqueous solution of glycerol. Can be stored on a rich agar medium with glucose for 3 months at +4°C.

Stability:

The stability of the inventive strains stored at 20 successive subcultures on agar YPD medium at a temperature of 28°C.

Pathogenicity: are not patientname.

The invention illyustriruetsya following examples are presented to confirm, but do not limit the amount of pritasari.

Examples.

Example 1. Design vector pPDX3-HUB

The structural gene of ubiquitin yeast that encodes a protein with sequence represented in SEQ ID NO 1 or 2, amplified in a PCR reaction using as the template the chromosomal DNA laboratory strain of S. cerevisiae Y618 [Kartaseva et al, 1996, Yeast, v.12, 1297-13]allocated according to the method of Sydoruk [Sidoruk et al., 2008 book of abstracts and reports on Topical issues of genetics, radiobiology and radioecology", Dubna, JINR, p.100]. Primers for amplification are N513 (5'-ataccatggaacatcatcatcatcatcatggaggcatgcagatcttcgtcaagactttga) and # 514 (5'-actggatccacctcttagccttagcacaac). The resulting amplification of the DNA fragment size 510 P.O. elute from agarose gel using the Qiagen kit (Qiagen, cat. No. 28706), treated with restrictase Ncol and BamHI and clone a split on the same site laboratory plasmids pUC18x-GAL1-Ncol, carrier Hindlll/Ncol fragment of DNA encoding the promoter region of the gene GAL1 yeast. S.cerevisiae. The result is the plasmid p101-25, containing the nucleotide sequence encoding the ubiquitin yeast S.cerevisiae, merged with the nucleotide sequence of the promoter region of the gene GAL1 yeast S.cerevisiae. Plasmid pi01-25 contains the website to know restrictase XhoI in polylinkers area behind the site of the BamHI cloning.

HindIII/XholI DNA fragment from plasmid p101-25, including the promotor region of the GAL1 gene and the nucleotide sequence encoding ubiquitin, clone lab vector pPDX3, DNA which is split according to the same sites. In the cloning receive vector pPDX3-HUB, which is used for gene cloning of recombinant proteins the web.

Example 2. Designed for the W expression vector pPDX3-HUB-1F9

Expression vector pPDX3-HUB-1F9 (Figure 3) produced by cloning BgllllXhol DNA fragment laboratory plasmid pUC21-1F9 size 3.6 TPN containing the gene of the protein 1F9, vector pPDX3-HUB, DNA which is split across sites BamHI and Xhol. In the cloning receive expression vector pPDX3-HUB-1F9, in which the structural gene of the protein 1F9 fused in the same reading frame with the structural gene coding for ubiquitin. The vector used for expression of the protein 1F9 in yeast S.cerevisiae.

Example 3. Construction of expression vector pPDX3-HUB-2E12

Expression vector pPDX3-HUB-2E12 receive as a result of cloning BgIII/XhoI DNA fragment laboratory plasmid pUC21-E size 4.2 TPN containing the gene of the protein A, vector pPDX3-HUB, DNA which is split across sites BamHI and Xhol. In the cloning receive expression vector pPDX3-HUB-2E12, in which the structural gene of the protein A fused in the same reading frame with the structural gene coding for ubiquitin. The vector used for expression of the protein A in yeast S.cerevisiae.

Example 4. Design vector pPDX3-SUMO.

Structural gene SMT3 yeast S.cerevisiae, encoding a protein with SUMO sequence represented in SEQ ID NO 3, amplified in the reaction Poland using as the template the chromosomal DNA laboratory strain S.cerevisiae as in example 1.

Amplify what the situation is carried out in two stages. First amplified two overlapping DNA fragments, what do the following pairs of primers:

Fragment 1 size 129 BP:

Amplificatoare fragments elute DNA from agarose gel and used for PCR-ligation. For this purpose, the mixture of fragments 1 and 2 are used as matrix for PCR, primers are used N450 and N452. The resulting PCR DNA fragment size 290 BP elute from agarose gel, treated with restrictase BglII and BamHI and clone into the BamHI site laboratory plasmids pUC18x-GALl-BamHI carrying HindIII/BamHI DNA fragment encoding the promoter region of the gene GAL1 yeast S.cerevisiae, containing the ATG codon and a BamHI site (underlined) in the sequence ATGCATGGATCC. The result will be merged sequence of the gene SMT3 yeast S.cerevisiae and sequence that encodes a promoter region of a gene GAL1 yeast S.cerevisiae. The result is the plasmid R-18, in which the cloned gene SMT3 is sequenced.

The obtained plasmid R-18 contains a DNA fragment, in which the gene SMT3 yeast merged with the promotor region of the gene GAL1 yeast. In polylinkers part plasmids R-18 behind the site of the BamHI cloning site is recognition of restrictase XhoI. HindIII/XhoI DNA fragment from plasmid R-18, including the promotor region of the GAL1 gene and the cloned gene SMT3, clone laboratory is ornam vector pPDX3, DNA which is split according to the same sites. In the cloning receive vector pPDX3-SUMO, which is used for gene cloning of recombinant proteins the web.

Example 5. Construction of expression vector pPDX3-SUMO-1F9.

Expression vector pPDX3-SUMO-1F9 (Figure 4) is obtained cloned BglII/XhoI DNA fragment laboratory plasmid pUC21-1F9 size 3.6 TPN containing the gene of the protein 1F9, vector pPDX3-SUMO, DNA which is split across sites BamHI and XhoI. In the cloning receive expression vector pPDX3-HUB-1F9, in which the structural gene of the protein 1F9 fused in the same reading frame with the structural gene coding for ubiquitin. The vector used for expression of the protein 1F9 in yeast S.cerevisiae.

Example 6. Construction of strain SCR-702-1F9 - producer protein 1F9 (VKPM Y-3583).

Strain SCR-702-1F9 receive as a result of the transformation of the laboratory strain D702 expression vector pPDX3-HUB-1F9. To implement the transformation of cells of strain D702 pokasivaut for 18-24 hours at the temperature of 28°C on agar medium YPGE, of the following composition in wt.%: bactopeptone - 2, yeast extract - 1, baktagir - 2, ethanol, 2 - glycerol - 3, water - the rest. The transformation of the grown cells of strain D702 carried out according to the method of Ito et al. [Ito et al., 1983, J. Bacteriol., v.153, 163-168]. Transformants are selected based on their ability to grow on YPD medium following is the composition in wt.%: bactopeptone - 2, yeast extract - 1, glucose - 2, baktagir - 2, water - the rest. One of the obtained transformants called SCR-702-1F9.

Example 7. Construction of strain SCR-702-2E12 - producer protein E.

Strain SCR-702-2E12 receive as a result of the transformation of the laboratory strain D702 expression vector pPDX3-HUB-2E12. The transformation is carried out as in example 6. Strain SCR-702-2E12 deposited in Russian national Collection of Industrial Microorganisms as Saccharomyces cerevisiae strain VKPM Y-3584.

Example 8. Construction of strain D702-SUMO-1F9-producer protein 1F9.

Strain D702-SUMO-1F9 receive as a result of the transformation of the laboratory strain D702 expression vector pPDX3-SUMO-1F9. The transformation is carried out as in example 4 except that the use of plasmid pPDX3-SUMO-1F9.

Example 9. Analysis of expression of recombinant protein 1F9 and E in cells of Saccharomyces cerevisiae strains.

Cells of S. cerevisiae VKPM Y-3583, VKPM Y-3584 or D702-SUMO-1F9 cultured in flasks at 30°C on a rotary shaker at 250 rpm on a YPD liquid medium of the composition, in wt.%: bactopeptone - 2, yeast extract - 1, glucose - 2, water - the rest, sowing in the titer of 5×105- 5×106ml-1. Samples for assay, after 46 hours of culture growth. The final optical density of the culture is OD600=40-45. Cells are separated from the environment of the cultivation of precipitation by centrifugation PR is 10000 g for 1 min and used for subsequent analysis of protein expression 1F9 and A micromethod in vitro 1.5 ml. For this sediment cells suspended in buffer for destruction" (0.05 M phosphate, 2.5 mm EDTA, 5% glycerol) at the rate of 100 μl of buffer 100 ál of wet sediment cells. The destruction of cells is carried out using glass beads (d=0,45-0,65 mm) on a shaker for test tubes type "Vortex". To do this, 570 mg of beads mixed with 200 ál of cell suspension, the mixture is shaken at 0°C for 90 sec, to the contents of the test tubes add 250 ál of buffer for destruction" and shaking repeat for 60 seconds. In tubes make 500 μl of buffer for destruction", the contents of the tubes are mixed, after which the samples centrifuged for 10 min at 16000 g. The supernatant containing soluble proteins in yeast cells, using 1 M sodium acetate solution pH adjusted to 4.0 and the precipitated material is removed by centrifugation for 5 min at 16 Tyson/min; the supernatant is then heated at 65°C for 20 minutes and loose sediments of ballast proteins are removed by centrifugation; the resulting solution cialiswhat 40 minutes against 10 mm sodium acetate, pH of 4.0. The precipitated water-insoluble proteins are suspended in 750 μl of buffer for destruction"is transferred into a new tube and centrifuged for 15 min at 16000 g. The precipitate (100 μl)containing the target proteins are suspended in 400 µl buffer 6,5G" (6.5 M solution of guanidine hydrochloride or guanidine thiocyanate the buffer, containing 0.1 M of sodium phosphate, 0.01 M Tris-HCl, pH 6.5) and target proteins extracted overnight on a magnetic stirrer at a temperature of +4°C. Then the suspension is centrifuged for 15 min at 16000 g, the supernatant passed with him a target protein cialiswhat against 300 ml of 5 mm sodium acetate for 1.5 hours. The resulting sample is centrifuged for 15 min at 16000 g and the supernatant used for electrophoretic analysis of the level of production of the target protein.

Electrophoretic analysis of the target protein spend 12% SDS page-DDS-Na according to the standard procedure of laemmli's method [Laemmli, 1970, Nature, v.227, 680-685]. To analyze the solution is diluted to approximately 500 - 1000 times "sample buffer" (0.0625 M Tris-HCl, pH 6.8, 2 wt.% DDS-Na, 0.0025 wt.% Bromphenol blue). And boil in water bath for 5 minutes. Aliquots 3-15 μl applied to 12% SDS page and subjected to electrophoresis in a Bio Rad MiniPROTEAN until the dye front is at a distance of 1 cm from the end of the gel. The gels are washed in water and stained in 0.2% solution of Kumasi R-250 (Fermentas). Electrophoretic analysis shows that protein 1F9 and E accumulate in the fraction of water-insoluble proteins of yeast cells S. cerevisiae and does not contain component SUMO or ubiquitin (Figure 1 and 2). This is indicated by electrophoretic mobility of the analyzed proteins and the absence of gel protein bands corresponding to the higher mobility of the hybrid protein 1F9 and E, merged with what Elcom SUMO or ubiquinol.

Quantitative assessment of the purity of the preparations was carried out using the program "Videodensitometry Sorbfil 1.0", which stained gels after electrophoresis scanned, the resulting image was entered into the computer and an estimate of the amount of protein in the spot and on each track were determined using the program. As comparison standards used highly purified preparations of the proteins, known amount which was applied to the adjacent track in the same gel. The purity of the preparations of proteins, estimated in this way was 96% and above.

Example 10. Products protein 1F9 and E strains of Saccharomyces cerevisiae VKPM Y-3583 and VKPM Y-3584

To obtain the inoculum strains VKPM Y-3583 and VKPM Y-3584 grown in YPD medium on a rotary shaker at 250 rpm at a temperature of 28°C for 20-24 hours. 50 ml of inoculum used for the planting of a 3-liter fermentor Anglicon containing 950 ml of YPD medium. The fermentation is carried out at a temperature of 28°C, aeration 1 l/min and the stirring speed 1000 rpm After 24 hours after inoculation of the fermenter begin feeding environment of the cultivation of 50%glucose solution at the rate of 2 ml/h and set pH staterevenue culture at pH 6.8±0.1, using Podarok solution 10% sulfuric acid and 10% NaOH. The average total time of fermentation is 72 hours. According to the electro is eroticheskoe analysis of the products of protein 1F9 in these conditions is not less than 200 mg/l of culture fluid and protein products E in these conditions is not less than 100 mg/l of culture fluid.

Example 11. Isolation and purification of the recombinant protein 1F9 and E of the water-insoluble fraction of cells Saccharomyces cerevisiae

Isolation and purification of protein 1F9 and E of the water-insoluble fraction of cells of strains-producers of VKPM Y-3583 and VKPM Y-3583 performed using methods described Boguchan et al. [Bogush V.G. et al., 2001, Biotechnology, Vol.2, 11-22; Bogush V.G. et al., 2006, Biotechnology, V. 4, 3-12; Bogush V.G. et al., 2009, J. Neuroimmune Pharmacol., v.4, 17-27]. When increasing the biomass of the yeast S.cerevisiae 3-liter fermenter medium YPD without makeup in the presence of 2% glucose in the starting environment with one fermentation receive an average of 400-500 g wet cell biomass. 1 kg of the washed wet biomass suspended in the buffer to fracture and destroy cells with glass beads in running the mill for 1.5 hours, centrifuged, the resulting suspension and collect the precipitate. The extraction of the target protein from sediment carried out using a solution of 10% lithium chloride in 90%formic acid for 16-18 hours, followed by centrifugation. The precipitate discarded, and the supernatant is subjected to ultrafiltration followed by diafiltration through the membrane M50 for translation in 10 mm sodium acetate, pH 4.0 and removal of proteins of the host cell with a molecular weight below 50 kDa.

Final cleaning is performed using ion-exchange chromatography on national the military column HiPrep 16/10 SP FF (GE Healthcare) in the system FPLS. After passing the filtrate through a column and then washing the column with 10 mm Na-phosphate buffer, pH 7.0 and then with 10 mm sodium acetate buffer, pH 4,0, protein 1F9 and E elute column with 10%NaCl in the same buffer and identified by electrophoresis in 12% SDS page-DDS-Na, fraction of protein target unite, dialist against deionized water, frozen at-70C° C and freeze-dried. Liofilizovannye drug is a substance of white color, similar to wool.

Example 12. Characterization of protein 1F9 and A

For analysis of the obtained preparation of pure recombinant protein is dissolved a portion of the drug in 90%formic acid with 10%lithium chloride for at least 2 hours, dialist against deionized water (1-1 .5 hours) and analyzed by SDS electrophoresis in 12% SDS page. The presence of a single band in the gel, corresponding molekulyarnomu the mass of the recombinant protein, confirms the homogeneity of the final product. The obtained preparations of both proteins are characterized by the extinction coefficient which is approximately equal to 0.48±0,02 ND280/mg. This value corresponds to theoretically calculated on the basis of amino acid composition of these proteins (0,49 ND280/m), and indicates the high purity of the obtained products.

For analysis of the ability cleared the s recombinant proteins, web of spider-crporate to form supramolecular structures of different types of these proteins were tested for ability to form is not soluble in water thread. Threads get as a result of spinning (spinning) of concentrated protein solution through a narrow opening. For this purpose a portion of the purified lyophilized preparation of protein dissolved in 90%formic acid with 10%lithium chloride for at least 2 hours, deleteroute against deionized water for 1-1,5 hours, nerastvorim material removed by centrifugation. The protein solution is passed through a specially constructed micromineral with an inner diameter of about 50 μm with a speed of 5-10 μl/min in the coagulation bath with 96% ethanol. This forms a water-insoluble thread that freely falls to the bottom of the vessel. Newly formed artificial filament in a vessel with ethanol presents on Fig. The newly created thread is kept in a vessel with 96% alcohol for 20 minutes, then the maximum stretch in 75%ethanol, annealed, soak in deionized water and dried in air. The filaments are subjected to all stages of the impact, characterized by the relative values of the breaking load of 10-15 CN/Tex (13 MPa).

The results show that for the proposed method of producing recombinant proteins, web of spider-crporate a significant increase in the yield of recombinant proteins, and the resulting recombinant proteins are characterized by you the Oka purity and physicochemical properties, characteristic of the natural proteins of the web. The proposed method of producing recombinant protein wide web allows you to obtain the recombinant protein with a very high degree of purification on an industrial scale, to develop ways of micropreemie to obtain on the basis of artificial fibers, and methods of forming the films, hydrogels and microgels and microcapsules based on recombinant proteins webs for use in biotechnology, medicine, cosmetology, automotive, aerospace and other fields.

1. A method of obtaining a protein of web of spider-crporate in the cells of the yeast Saccharomyces cerevisiae, providing for the construction of expression vector containing a DNA sequence encoding a recombinant protein of web of spider-crporate, merged with a sequence that encodes a ubiquitin or ubiquitin-like protein SUMO yeast Saccharomyces cerevisia, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, transformation of yeast cells obtained expression vector and expression in transformed cells protein web of spider-crporate, and ubiquitin has a sequence selected from the group of SEQ ID NO: 1 or 2, and SUMO protein, has the sequence of SEQ ID NO: 3.

2. The method according to claim 1, characterized in that the use of the expression vector, which contains a DNA sequence encoding a recombinant protein of web of spider-crporate, consensus sequences which originate from proteins frame is large and/or small ampoule glands or proteins hunting thread.

3. The method according to claim 1, characterized in that use expression vector that contains a DNA sequence encoding a recombinant protein of web of spider-crporate, consensus sequences which originate from proteins frame large sealed gland of Nephila clavipes and/or Nephila madagascariensis.

4. The method according to claim 1, characterized in that use perepilichny vector pPDX3-HUB-2E12, including scope for replication initiation of endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein A frame thread of spider-crporate Nephila madagascariensis, merged with a sequence that encodes a ubiquitin Saccharomyces cerevisiae.

5. The method according to claim 1, characterized in that use perepilichny vector pPDX3-HUB-1F9, including scope for replication initiation of endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein 1F9 frame thread of spider crporate Nephila clavipes, merged with a sequence that encodes a ubiquitin Saccharomyces cerevisiae.

6. The method according to claim 1, characterized in that use perepilichny vector pPDX3-SUMO-1F9, including scope for replication initiation of endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes, merged with a sequence that encodes a protein SUMO Saccharomyces cerevisiae.

7. Protein comprising the recombinant protein of web of spider-crporate and ubiquitin or ubiquitin-like protein SUMO yeast Saccharomyces cerevisiae, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and the sequence of the recombinant protein of the web includes a consensus sequence that occur from repeated sequences of proteins of frame threads MaSp1 and/or MaSp2 large sealed gland, protein MiSp1 and/or MiSp2 small sealed gland, or protein Flag hunting thread of spider-crporate, the sequence encoding the ubiquitin presented in SEQ ID NO: 1 or 2, and the sequence encoding a ubiquitin-like protein represented in SEQ ID NO: 3.

8. Fused protein according to claim 7, in which the sequence of the recombinant protein the web of spider-crporate includes a consensus sequence derived from repetitive sequences of proteins of frame threads MaSp1 and MaSp2 large sealed gland of Nephila clavipes and Nephila madagascariensis and selected from the group:
AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGLGGQG
AGQGAGASAAAAGGAGQGGYGGLGSQG
AGRGGLGGQGAGAVAAAAAGGAGQGGYGGLGSQG
AGRGGQGAGAAAAAAGGAGQRGYGGLGNQG
GPGGYGPGQQGPGAAAAASA
GRGPGGYGPGQQGPGGSGAAAAAA
GSGPGGYGPGQQGPGGPGAAAAAAA
GRGPGGYGPGQQGPGQQGPGGSGAAAAAA
GRGPGGYGPGQQGPGGPGAAAAAA
GPGGYGPGQQGPGAAAAAAA
GSGAGGYGPGQQGPGGPGAAAAAA
GSGPGGYGPGQQGPGGSSAAAAAA
GSGPGGYGPGQQGPGGSGAAAAAAAA
GRGPGGYGQGQQGPGGPGAAAAAA.

9. Fused protein according to claim 7, characterized in that it contains the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes and ubiquitin yeast Saccharomyces cerevisiae and has the sequence SEQ ID NO: 1.

10. Fused protein according to claim 7, characterized in that it contains the sequence of the recombinant protein frame thread A spider crporate Nephila madagascariensis and ubiquitin yeast Saccharomyces cerevisiae and has the sequence SEQ ID NO: 2.

11. Fused protein according to claim 7, characterized in that it contains the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes and SUMO protein of yeast Saccharomyces cerevisiae and has the sequence SEQ ID NO: 3.

12. Recombinant DNA encoding a fused protein according to claim 7, comprising a sequence of the recombinant protein the web of spider-crporate and ubiquitin or ubiquitin-like protein SUMO yeast Saccharomyces cerevisiae, which is in the composition of the fused protein N-terminal position relative to the recombinant protein of the web, and the sequence of the recombinant protein of the web includes consensus sequences that originate from Cycling the working sequences of proteins of frame threads MaSp1 and/or MaSp2 large sealed gland, protein MiSp1 and/or MiSp2 small ampoule cancer or protein Flag hunting thread of spider-crporate, the sequence encoding the ubiquitin presented in SEQ ID NO: 1 or 2, and a sequence encoding a ubiquitin-like protein represented in SEQ ID NO: 3.

13. Recombinant DNA according to item 12, where the consensus sequence of recombinant analog of the protein of web of spider-crporate represent sequences derived from repetitive sequences of proteins of frame threads MaSp1 and MaSp2 large sealed gland of Nephila clavipes and Nephila madagascariensis and selected from the group:
AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGGAGQGGLGGQG
AGQGAGASAAAAGGAGQGGYGGLGSQG
AGRGGLGGQGAGAVAAAAAGGAGQGGYGGLGSQG
AGRGGQGAGAAAAAAGGAGQRGYGGLGNQG
GPGGYGPGQQGPGAAAAASA
GRGPGGYGPGQQGPGGSGAAAAAA
GSGPGGYGPGQQGPGGPGAAAAAAA
GRGPGGYGPGQQGPGQQGPGGSGAAAAAA
GRGPGGYGPGQQGPGGPGAAAAAA
GPGGYGPGQQGPGAAAAAAA
GSGAGGYGPGQQGPGGPGAAAAAA
GSGPGGYGPGQQGPGGSSAAAAAA
GSGPGGYGPGQQGPGGSGAAAAAAAA
GRGPGGYGQGQQGPGGPGAAAAAA.

14. Recombinant DNA according to item 12, encoding a protein containing the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes and ubiquitin yeast Saccharomyces cerevisiae and having the sequence of SEQ ID NO:1.

15. Recombinant DNA according to item 12, encoding a protein containing the sequence of the recombinant protein A frame thread of spider-crporate Nephila madagascariensis and ubiquitin yeast Saccharomyces cerevisiae and having the sequence of SEQ ID NO:2.

16. Recombinant DNA according to item 12, the coding is based on protein, containing the sequence of the recombinant protein 1F9 frame thread of spider-crporate Nephila clavipes and SUMO protein of yeast Saccharomyces cerevisiae and having the sequence of SEQ ID NO:3.

17. The expression vector containing the recombinant DNA according to item 12.

18. The expression vector for 17, including recombinant DNA, protein-coding web of spider-crporate, consensus sequences which occur from repetitive sequences, proteins frame large sealed gland of Nephila clavipes and/or Nephila madagascariensis, and the ubiquitin or ubiquitin-like protein SUMO yeast Saccharomyces cerevisiae.

19. The expression vector according to 17, representing perepilichny vector pPDX3-HUB-2E12 and containing an area of replication initiation endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant analogue of the protein E, merged with a sequence that encodes a ubiquitin Saccharomyces cerevisiae.

20. The expression vector according to 17, representing perepilichny vector pPDX3-HUB-1F9 and containing an area of replication initiation endogenous 2-micron plasmid of yeast promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant analogue of the protein 1F9, merged with a sequence that encodes a ubiquitin Saccharomyces cerevisiae.

21. The expression vector according to 17, representing perepilichny vector pPDX3-SUMO-1F9 include the s region of initiation of replication of endogenous 2-micron plasmid of yeast, the promotor region of the gene of the yeast GAL1, a DNA sequence encoding a recombinant protein 1F9, merged with a sequence that encodes a protein SUMO Saccharomyces cerevisiae.

22. A host cell of Saccharomyces cerevisiae producing recombinant protein frame thread web of spider-crporate transformed by the expression vector by 17.

23. The Saccharomyces cerevisiae strain VKPM Y-3583 producing recombinant protein 1F9 frame thread web of spider-crporate and transformed by the expression vector pPDX3-HUB-1F9.

24. The Saccharomyces cerevisiae strain VKPM Y-3584 producing recombinant protein A frame thread web of spider-crporate and transformed by the expression vector pPDX3-HUB-2E12.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing polyunsaturated fatty acids in seeds of transgenic plants. The method involves introducing into a plant, nucleic acids whose sequences code enzymes having Δ-6-desaturase, Δ-6-elongase, Δ-5-desaturase, Δ-5-elongase, Δ-4-desaturase and Δ-12-desaturase activity.

EFFECT: method increases content of polyunsaturated fatty acids in seeds of transgenic plants.

1 cl, 33 dwg, 24 tbl, 61 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology. The invention describes a yeast cell which contains an interference DNA construct. The construct comprises: a reporter gene under control of a first promoter; one or more in induced promoters called interference promoters selected and fixed in such a way that their activation induces transcriptional interference of the first promoter leading to a detectable decrease in expression of the reporter gene. The cell expresses: a first chimeric protein (Y-AD) formed by a transcription activation domain (AD) fused with a protein Y capable of interacting with at least a partner protein X; a second chimeric protein (X-DBD) formed by a first DNA-binding domain (DBD) fused with a second domain formed by protein X capable of interacting with protein Y, wherein the interaction of the two chimeric proteins X-DBD and Y-AD leads to formation of a functional transcription factor which activates the interference promoter(s). The invention discloses a method of intensifying a compound which inhibits interaction of the first protein X with the second protein Y.

EFFECT: invention enables detection of interruption of protein-protein interaction, as well as identification of alleles lacking on the level of interaction of proteins involved in the protein-protein interactions.

27 cl, 11 dwg, 4 tbl, 11 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is a Pachia pastoris PS 108 (pChIG) yeast strain, which is a producer of chicken interferon-gamma transformed by plasmid pChIG. Such plasmid pChIG, which facilitates biosynthesis of chicken interferon-gamma, has size of 8.19 thousand base pairs and consists of the following elements: - EcoRI-BamHI - a fragment of plasmid DNA of bifunctional bacterial-yeast vector pPIC9 with size of 7.75 thousand base pairs, - Bglll-EcoRI - a fragment with size of 0.44 thousand base pairs, which contains the coding part of the chicken interferon-gamma gene except the signal peptide coding region. The chicken interferon-gamma gene is obtained through a reverse polymerase chain reaction, whose matrix is RNA obtained from chicken peripheral blood mononuclear cells, induced by human recombinant interleukin-2 Roncoleikin ®.

EFFECT: invention enables obtaining chicken interferon-gamma with high efficiency.

3 cl, 2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: there is offered method for identification and/or verification of inhibitors of receptor tyrosine kinases that involves application of a new test system which represents a yeast host cell containing an expression vector including a nucleic acid sequence that encodes fused protein essentially consisting of a complete cytoplasmic part of analysed receptor tyrosine kinase and the dimerisation domain and, if necessary, in addition including anchoring sequence for fused protein in a membrane wherein expression of fused protein conduces to termination of cell proliferation. The method provides production of specified host cells being in contact with a candidate compound and identification of inhibitors of tested tyrosine kinase activity as a result of cultivation on the assumption that inhibition of tyrosine kinase activity with a candidate compound causes restoration of proliferation process.

EFFECT: prospected application of the invention is related to development of selective therapeutic, including anticancer, agents.

13 cl, 5 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, more specifically to use of transformed cells Saccharomyces cerevisiae, which belong to a strain used in wine making, capable of high urea degradation in conditions of fermentation of grape juice, compared untransformed cells of the said strain. The cell is transformed by recombinant nucleic acid, which contains a sequence, which includes an open reading frame, homologic open reading frame DUR1,2 and a coding protein, with urea-degrading fermentation activity, and a promoter, suitable for mediating expression of a protein with urea-degrading fermentation activity, in conditions of fermentation of grape juice.

EFFECT: invention allows for obtaining wine, which cannot be distinguished from its organoleptic properties from wine obtained using untransformed cells.

13 cl, 7 dwg, 1 tbl, 1 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to genetic engineering and bioengineering and can be used for production of recombinant human mechanodependent growth factor (MGF). Recombinant human mechanodependent growth factor is produced by cultivation of yeast cells containing plasmid which provides expression of protein secretor, and liberation of end product from culture fluid. Plasmid providing expression of human mechanodependent growth factor secretor contains expression cartridge including GAL1 gene promotor and termination region of yeast CYC1 gene transcription, pre- MFα1 gene region and DNA sequence, coding end protein. This plasmid is composed on the basis of plasmid pKX. Besides for production of human mechanodependent growth factor, strain Saccharomyces cerevisiae YBS618/pKX-MGF - producer of specified produced is also used.

EFFECT: expression of human mechanodependent growth factor secretor in yeast.

6 cl, 2 dwg, 1 tbl, 7 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.

EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.

2 cl, 7 dwg

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to yeast strain Pichia pastoris PS106(pHIG) that represents a producer of human immune interferon, to recombinant plasmid used for transformation of this strain, and to a method for design of such plasmid. Invention provides preparing a recombinant gamma-interferon that can be secreted into cultural fluid in industrial scales. Advantage of invention involves the development of plasmid able to produce recombinant gamma-interferon for large-scale production.

EFFECT: valuable properties of yeast strain.

3 cl, 2 dwg, 3 ex

FIELD: biotechnology, molecular biology, genetic engineering.

SUBSTANCE: method involves transformation of the strain yeast Pichia pastoris PS99 (his4 pep4::PHO85) with the constructed plasmid pPIC9HabIL-2 and preparing the strain Pichia pastoris PS107(pPIC9HabIL-2) that represents a producer of hybrid protein consisting of human plasma blood albumin and human IL-2. Invention provides preparing highly stable hybrid protein consisting of human plasma blood albumin and human IL-2 by simplified technology Invention can be used in preparing hybrid proteins preparations.

EFFECT: improved preparing method, valuable properties of strain and hybrid protein.

4 cl, 2 dwg, 5 ex

FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.

SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.

EFFECT: method for large-scale production of polyunsaturated fatty acid.

15 cl, 4 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and virology. Compositions and methods used to induce immune response against the influenza virus in canines using novel strains, polynucleotides or polypeptides thereof are described. The invention can be used in veterinary.

EFFECT: versions of an influenza virus, which can infect canines and cause a respiratory disease in canines are disclosed.

38 cl, 14 dwg, 25 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: there is constructed a recombinant plasmid DNA pAP271 containing a rhFVII protein gene, a MAR matrix attachment region of an avian lysozyme gene, CMV virus transcription amplifier, a promoter of translational factor of human EF-1α elongation, an internal site of translational initiation of encephalomyocarditis IRES virus, a mouse DHFR gene, a SV40 virus polyadenylation signal, a cartridge comprising all elements required for aminoglycoside-3'-phosphotransferase (APH) gene expression, a cartridge for expression in bacterial cells of β-lactamase gene, as well as unique recognition sites of the following restriction endonucleases: Agel (850 base pairs), BbvCI (1657 base pairs), BmgBI (4202 base pairs), BssHII (6672 base pairs), Eco47III (11269 base pairs), EcoRI (10929 base pairs), EcoRV (11863 base pairs), Fsel (1455 base pairs), NotI (4812 base pairs), RsrII (6790 base pairs), Sbfl (2330 base pairs), Sfil (6027 base pairs), Tthllll (6390 base pairs), Xcml (2404 base pairs).

EFFECT: presented invention provides producing stably high-yield recombinant human blood coagulability factor VII.

2 cl, 4 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: composition contains an effective amount of a full length hepatocyte growth factor (flHGF) and a deletion version of hepatocyte growth factor (dHGF), or one or more polynucleotides coding said isoforms.

EFFECT: invention provides effective treatment and preventing of cardiac diseases in a subject, and also activation of endothelial cell growth in a blood vessel.

39 cl, 20 dwg, 4 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: microorganism, which codes antigens and protein toxins, contains a first component, which is at least one nucleotide sequence coding at least at least one complete or partial antigen of at least one wild-type or mutated protein, a second component which is at least one nucleotide sequence coding for at least one protein toxin and/or at least one protein toxin subunit, a third component consisting of at least a first subcomponent which is at least one nucleotide sequence coding at least one transport system which enables the expression of the first and second components on the outer surface of the microorganism and/or enables the secretion of the expression products of the first and second components, and/or coding at least one signal sequence which enables the secretion of the expression products of the first and second components, and/or optionally, from the second subcomponent, which is at least one nucleotide sequence coding at least one protein for lysing the microorganism in the cytosol of mammalian cells and for intracellularly releasing plasmids or expression vectors, which are contained in the lysed microorganism; and a fourth component which is at least one nucleotide sequence for at least one activation sequence for the expression of one or more of the first, second and third components, wherein said activation sequence can be activated in the microorganism and/or is tissue cell-specific, tumour cell-specific, macrophage-specific, dendrite-specific, lymphocyte-specific, function-specific or non-cell-specific, wherein any of the first, second, third or fourth components, present in the microorganism more than once, are independently identical or different, wherein the first and second components are different from each other. Also disclosed are a medicinal agent for stimulating immune response and a pharmaceutical composition based on said microorganism, methods of obtaining said microorganism, the corresponding expression plasmid and expression vector for obtaining said microorganism.

EFFECT: invention enables to obtain novel antitumour vaccines, which induce a strong systemic cellular immune system response, which increases effectiveness of antitumour therapy.

21 cl, 22 dwg, 8 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and immunology. There are offered: a monoclonal antibody or its fragment specifically bound with GDF8 and not bound with BMP11, a polynucleotide coding it, an expression vector and a host cell for antibody expression. There are studied: a method for producing the GDF8-specific antibody, a method for GDF8 presence test, a method of treating a GDF8-associated disorder.

EFFECT: use of the invention provides the new GDF8-specific antibodies that can find further application in the therapy of the GDF8-mediated diseases.

22 cl, 33 dwg, 8 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a system of modulating expression of a nuclear receptor-based inducible gene, which includes two expression cassettes, where the first cassette contains a polynucleotide which codes a first polypeptide consisting of a) a DNA-binding domain represented by an ecdysone receptor domain (EcR), GAL4 or LexA and b) a ligand-binding domain (LBD) of EcR containing at least a mutation in a position corresponding to position 132 in the ecdysone receptor sequence Chotistoneura fumiferctna (CfEcR), which increases sensitivity of the system to diacyl hydrazine ligands, and the second expression cassette contains a polynucleotide which codes a second polypeptide consisting of a) a transactivation domain represented by a transactivation domain of EcR, VP16, an acidic activator B42 or p65, and b) a second LBD which is a nuclear receptor domain capable of dimerisation with a first mutant LBD, preferably a ligand-binding domain of retinoid X receptor.

EFFECT: use of present invention enables to control expression of recombinant proteins with synthetic ligands and, consequently, obtain the desired products in any type of animal and plant host cells.

12 cl, 14 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: what is offered is an antibody or its antigen-binding fragment which specifically coupling hlL-4R with KD less than 200-pM measured with using surface plasmon resonance. What is described is a recovered nucleic acid molecule coding the antibody, and a based vector for producing the antibody. There are disclosed a host-vector system for producing the antibody or its antigen-binding fragment, and a method for producing the substances stated above with using such system. What is disclosed is using the antibody or antigen-binding fragment for preparing a drug for relieving (inhibiting) hlL-4R mediated diseases. What is disclosed is a composition on the basis of the antibody or antigen-binding fragment to be used in a method for treating a hlL-4R mediated disease or disorder in humans.

EFFECT: inventions can find application in therapy of the hlL-4R mediated diseases.

15 cl, 3 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is described is fatty acid synthetase of a sequence presented in the description. What is specified is a polynucleotide coding such synthetase. What is presented is a vector containing such polynucleotide. Besides, what is also described is a transformed microbial organism containing such vector or polynucleotide. The transformed microbial organism is used to produce fatty acids. What is offered is a method for producing a lipid or a fatty acid that involves transformant cell culture followed by recovering the lipid or the fatty acid from such cells. What is described is a method for assessment of ability to form fatty acids based on the measurement of an expression level of fatty acid synthetase having a nucleotide sequence of specified polynucleotide. Also, there is presented a method for selection of microbial lipid-producing organisms that is based on the comparison of the expression levels of specified gene in a reference and tested microbial organism.

EFFECT: invention provides producing high-yield fatty acids.

17 cl, 1 dwg, 2 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: the invention relates to biotechnology and immunogenic compositions containing plasmid for expression of membrane antigen of hepatitis B virus (HBsAg). The said plasmid includes: (1) the sequence which encodes HBsAg, which has an aminoacid sequence SEQ ID NO:3; (2) a promoter of the glyceralgehyde-3-phosphate dehydrogenase, positioned above, to control the expression of the sequence which encodes HBsAg, and the promoter contains a gene sequence SEQ ID NO:1; and (3) the transcription terminator ARG3, positioned below the sequence which encodes HBsAg, where the terminator includes SEQ ID NO:8.

EFFECT: HBsAg can be purified and used to produce vaccines and, in particular, in production of monovalent and combined vaccines.

40 cl

FIELD: immunology and bioengineering.

SUBSTANCE: present invention refers to immunology and bioengineering. The variants of an antisubstance that is specific in relation to at least one globulomer Aβ(20-42) have been suggested. Each of the variants is characterized by the fact that it includes VH and VL parts; each of these parts contains three corresponding CDR. The antisubstance antigen-binding section has been revealed. They described: a coding nucleic acid and the vector that contains it, and a host cell that bears the vector that are used for the antisubstance production. The way of antisubstance production with the use of a cell has been discovered. The suggested inventions can find their application in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

EFFECT: antosubstances that can be used in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

10 cl, 28 dwg, 9 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a system of modulating expression of a nuclear receptor-based inducible gene, which includes two expression cassettes, where the first cassette contains a polynucleotide which codes a first polypeptide consisting of a) a DNA-binding domain represented by an ecdysone receptor domain (EcR), GAL4 or LexA and b) a ligand-binding domain (LBD) of EcR containing at least a mutation in a position corresponding to position 132 in the ecdysone receptor sequence Chotistoneura fumiferctna (CfEcR), which increases sensitivity of the system to diacyl hydrazine ligands, and the second expression cassette contains a polynucleotide which codes a second polypeptide consisting of a) a transactivation domain represented by a transactivation domain of EcR, VP16, an acidic activator B42 or p65, and b) a second LBD which is a nuclear receptor domain capable of dimerisation with a first mutant LBD, preferably a ligand-binding domain of retinoid X receptor.

EFFECT: use of present invention enables to control expression of recombinant proteins with synthetic ligands and, consequently, obtain the desired products in any type of animal and plant host cells.

12 cl, 14 tbl, 10 ex

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