Recombinant phistevtsib0821 plasmid; escherichia coli rosetta(de3)/phistevtsib0821 strain transformed by above said plasmid, and method for obtaining recombinant tsib_0821 prolidase

FIELD: biotechnologies.

SUBSTANCE: invention refers to biotechnology and gene engineering and represents recombinant pHisTevTSIB0821 plasmid for expression in Escherichia coli cells of TSIB_0821 prolidase from Thermococcus sibiricus archean. The proposed plasmid includes NdeI/SalI-fragment of pET-22b(+) (Novagen) plasmid and a DNA fragment with the size of 1196 pairs of bases, which contains a fused gene consisting of the following structural elements: nucleotide sequence coding 6-histidine tag, nucleotide sequence coding the site of recognition/decomposition of TEV protease, and nucleotide sequence of TSIB_0821 gene, which are connected so that at their biosynthesis in E. coli cells a continuous reading frame can be maintained. E. coli Rosetta(DE3) strain is obtained, which is transformed with the above plasmid, - a producer of chimeric protein including amino acid sequence of TSIB_0821 prolidase, fused on N-end with the 6-histidine tag and the site of recognition/decomposition of TEV protease. A growth and induction method of a producer strain and a method for separation and cleaning from the obtained biomass of functionally active recombinant TSIB_0821 prolidase including the following technological process: two metal affine chromatographies, gel filtration, TEV protease treatment, dialysis, and concentration, have been developed.

EFFECT: invention allows obtaining recombinant prolidase that is similar as much as possible as to structure to its natural equivalent with high and stable yield, level of cleaning and functional activity.

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The technical field to which the invention relates.

The present invention relates to biotechnology, in particular genetic engineering, and relates to a method of obtaining functionally active recombinant prolidase thermostable archaea Thermococcus sibiricus (TSIB_0821), and recombinant plasmids and transformed her Escherichia coli to produce recombinant prolidase TSIB_0821. The invention allows to produce functionally active recombinant prolidase for use in biotechnological processes, including those carried out at elevated temperatures and pH, as well as a model for studying the molecular mechanisms for thermostability.

The level of technology

Prolidase (polyspecificity dipeptidase, emidodapcinia, Y.S. 3.4.13.9., hereinafter referred to as prolidase) - enzyme gidrolizuemye dipeptides in which Proline is located in C-terminal position (XAA-Pro or X-Pro) [Cunningham, D. & O'connor, C. Proline specific peptidases // Biochim Biophys Acta. -1997. -Vol. 1343. -P. 160-186. and Lowther, W. & Matthews, B. Metalloaminopeptidases: common functional themes in disparate structural surroundings // Chem Rev. -2002. -Vol. 102. - P. 4581-4608]. Prolidase found in eukaryotes, bacteria and archaea and are extremely promising enzymes for use in medicine and biotechnology.

In mammals, prolidase participate in the final stages of catabolism, the breaking down of molecules rich in what Rolin, first of all collagen [Surazynski, A., Miltyk, W., Palka, J. & Phang, J. M. Prolidase-dependent regulation of collagen biosynthesis // Amino Acids. -2008. -Vol. 35. -P. 731-738]. In humans, deficiency of prolidase causes the development of a number of diseases of the connective tissue [Viglio, S., Annovazzi, L., Conti, B., Genta, I., Perugini, P., Zanone, C., Casado, C., Cetta, G. & ladarola, P. The role of emerging techniques in the investigation ofprolidase deficiency = MKD: from diagnosis to the development of a possible therapeutical approach // J Chromatogr In Analyt Technol Biomed Life Sci. -2006. -Vol. 832. -P. 1-8], against which as a substitution therapy using recombinant enzymes [Patent No. W002067967. -2002, Patent No. KR20050090120. - 2005, Patent No. US2006134088. -2006, Patent No. W02007110767. -2007., Colonna, C., Conti, B" Perugini, P., Pavanetto, F., Modena, I., Dorati, R., ladarola, P. & Genta, I. Ex vivo evaluation ofprolidase loaded chitosan nanoparticles for the enzyme replacement therapy // Eur J Pharm. Biopharm. -2008. -Vol. 70. -P. 58-65. and Colonna, S., Conti, B., Perugini, P., Pavanetto, F., Modena, I., Dorati, R., ladarola, P. & Genta, I. Site-directed PEGylation as successful approach to improve the enzyme replacement in the case ofprolidase // Int J Pharm. -2008. -Vol. 358. -P. 230-237]. At the same time, the increased level prolidase activity is a diagnostic marker of some cancers [Kama, E., Surazynski, A. & Palka, J. Collagen metabolism disturbances are accompanied by an increase in prolidase activity in lung carcinoma planoepitheliale // Int J Exp Pathol. -2000. -Vol. 81. -P. 341-347], for targeted therapy which create polysterene inactivated forms of the drugs that become active anticancer drugs after splitting prolidase [Patent W09745117. -1997, Mittal, S, Song, X., Vg, C. S. & Amidon, G. L. Proline prodrug of melphalan targeted to prolidase, a prodrug activating enzyme overexpressed in melanoma // Pharm Res. -2007. -Vol. 24.-P.1290-1298].

Prolidase bacteria and archaea are also involved in the degradation of proteins and peptides by regulating the recycling of Proline [Gonzales, T. & Robert-Baudouy, J. Bacterial aminopeptidases:

properties and functions // FEMS Environ. Rev. -1996. -Vol. 18. -P. 319-344. and Ghosh, M., Grunden, A. M., Dunn, D. M., Weiss, R. & Adams, M. W. Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus // J Bacteriol. -1998. -Vol. 180. -P. 4781-4789]. They are used to remove bitterness during the fermentation of cheese and other food industries [Courtin, P., Nardi, M., Wegmann, U, Joutsjoki, V., Ogier, J. C., Gripon, J. C, Palva, A., Heinrich, B. & Monnet, V. Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases // International Dairy Journal. -2002. -P. 447-454, Patent No. JP2008I78393. -2008, Patent No. US2010317736. -2010.J. in Addition, the majority of the studied today prolidase, primarily prolidase from thermophilic archaea active against toxic organophosphorus molecules, members of the nerve gases and pesticides [Vyas, N.K, Nickitenko, A., Rastogi, V. K., Shah, S. S. & Quiocho, F. A. Structural insights into the dual activities of the nerve agent degrading organophosphate anhydrolase/prolidase // Biochemistry. -2010. -Vol. 49. -P. 547-559. and Theriot, C.M., Tove, S. R. & Grunden, A. M. (eds.) Biotechnological Applications of Recombinant Microbial Prolidases // New York Academic Press. -2009], so high research activity is observed in the development of technologies aimed at the use the of prolidase as biosensors for the detection of toxic substances of this class [Simonian, A. L., Grimsley, J. K., Flounders, A. W, Shoeniger, J. S., Cheng, T.-C., DeFrank, J. J. & Wild, J. R. Enzyme-based biosensor for the direct detection of fluorine-containing organophosphates // Analytica Chimica Acta. -2001. -P. 15-23], means of biodecontamination [Cheng, T.C. & DeFrank, J. J. (eds.) Hydrolysis of organophosphorus compounds by bacterial prolidases // Dordrecht: Kluwer Academic Publishers. -2000] or components antidotes [Petrikovics, I., Mcguinn, W. D., Sylvester, D., Yuzapavik, P., Jiang, J., Way, J. L., Papahadjopoulos, D., Hong, K., Yin, R., Cheng, T. C. & DeFrank, J. J. In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism // Drug Deliv. -2000. -Vol. 7. -P. 83-89].

Today is characterized by slightly more than 10 prolidase from different organisms, which are listed in the review [R. Semcer and A. Grunden. Prolidase function in proline metabolism and its medical and biotechnological applications // J. Appl. Environ. -2012. doi: 10.1111/j.1365-2672.2012.05310]. Prospects search and comprehensive study of new enzymes of this class, especially from extremophilic archaea and cloning of the corresponding gene and preparation of recombinant proteases, including directed-modified by the methods of genetic engineering properties, evidenced by publications in periodicals and scientific journals and patent literature:

Patent No. US6309868. Cloning of the prolyl-dipeptidyl-peptidase from Aspergillus oryzae.-2001.

Patent No. US2003186420. Prolidase and its gene and method for producing prolidase. -2003.

Maher, M. J., Ghosh, M., Grunden, A. M., Menon, A. L., Adams, M. W., Freeman, H. C. & Guss, J. M. Structure of the prolidase from Pyrococcus furiosus // Biochemistry. -2004. -Vol. 43, -P. 2771-2783.

Patent No. JP2004105091. Heat-resistant poline-containing dipeptidase. -2004. Du, X., Tove, S., Kast-Hutcheson, K. & Grunden, A. M. Characterization of the dinuclear

metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants // FEBS Lett. -

2005. -Vol. 579. -P. 6140-6146.

Lupi, A., Delia Torie, S Campari, E., Tenni, R., Cetta, G, Rossi, A. & Forlino, A. Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies // FEBS J.-2006. -Vol. 273. -P. 5466-5478.

Patent No. JP3819454. New proline dipeptidase and hydrolysis of protein and peptide using the proline dipeptidase. -2006.

Yang, S.I. & Tanaka, T. Characterization of recombinant prolidase from Lactococcus lactis changes in substrate specificity by metal cations, and allosteric behavior of the peptidase //

FEBS J.-2008. -Vol. 275. -P. 271-280.

Besio, R., Alleva, S., Forlino, A., Lupi, A., Meneghini, C., Minicozzi, V., Profumo, A., Stellato, F., Tenni, R. & Morante, S. Identifying the structure of the active sites of human recombinant prolidase // Eur Biophys J.-2010. -Vol. 39. -P. 935-945.

Theriot, C. M., Tove, S. R. & Grunden, A. M. Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii // Appi Environ Biotechnol. -2010. -Vol. 86. -P. 177-188.

Theriot, C. M., Du, X., Tove, S. R. & Grunden, A. M. Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures // Appi Environ Biotechnol, -2010. -Vol. 87. -P. 1715-1726.

Theriot, C. M., Semcer, R. L., Shah, S. S. & Grunden, A. M. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures // Archaea. -2011. -Vol. 565127.

The above works demonstrate that for better use of the biotechnological potential of prolidase need to combine two strategies: firstly, to explore new enzymes, second, directed optimizion the te properties of natural enzymes according to the requirements of the process. Of particular interest in this regard are prolidase from extremophilic microorganisms, as they possess a wide range of unique properties, namely, capable of working in extreme conditions: high temperature, acidic or alkaline pH, high ionic strength, the presence of high concentrations of detergents. Therefore, to search for genes encoding new prolidase selected genome of the hyperthermophilic archaea Thermococcus Sibiricus MM739 living in hydrothermal vents and underground oil reservoirs in Western Siberia at a temperature of from 40°C. to 88°C. Analysis decoded the genome of T. Sibiricus indicates the presence of a gene TSIB 0821 (GenBank accession number ACS89882.1), encoding a protein similar to previously studied thermophilic protease from Pyrococcus horikoshii OT3 (52% homology).

As a prototype of the selected means of obtaining prolidase TSIB_0821, fused to the N-end with the sequence MRGSHHHHHHGS [Trofimov, A. A., Slutskaya, E. A., Polyakov, K. M., Dorovatovskiic, P. V., Gumerov, V. M., Popov, V. O. Influence of intermolecular contacts on the structure ofrecombinant prolidase from Thermococcus sibiricus // Acta Crystallogr Sect F Struct Biol Cryst Commun. -2012]. This method involves amplification of the gene TSIB_0821 (GenBank accession number ACS89882.1) on the matrix of genomic DNA of T. Sibiricus using polymerase chain reaction, its cloning in expressing vector pQE80L (Qiagen), obtaining an Escherichia coli strain Rosetta-gami (DE3) (Novagen)transformed with plasmid pQE80L_TSIB0821, capacity cells ø the Amma-producer, induction of expression and allocation of biomass and recombinant protein. The disadvantage of this method is the impossibility of removal from N-Terminus recombinant prolidase TSIB_0821 amino acid sequence sntihistamine tag, which, according to x-ray diffraction analysis of the fused protein MRGSHHHHHHGS-TSIB_0821, actively participates in the formation of spatial packing of the polypeptide, negatively affecting its functional activity and resistance to mechanical and thermal influences, making a recombinant prolidase TSIB_0821 obtained by this method unsuitable for use in biotechnological processes.

Disclosure of inventions

Technical challenge and, accordingly, the technical result of the invention is to develop a simple and environmentally friendly method of producing functionally active recombinant prolidase TSIB_0821 as close as possible in structure to its natural counterpart.

The problem is solved by the proposed method comprising the construction of plasmids pHisTevTSIB0821 that encodes the synthesis of recombinant prolidase TSIB_0821, fused to the N-end with the sequence sntihistamine tag and website recognition/cleavage protease TEV, on the basis of this plasmid strains E. coli Rosetta(DE3)/pHisTevTSIB0821 producer recombine the Noi prolidase TSIB_0821 in the form of a fused protein and the development of isolation and purification of recombinant prolidase TSIB_0821, which provides separation of the N-terminal amino acid sequence sntihistamine tag from the target polypeptide.

The technical result of the claimed invention is the preparation of recombinant prolidase TSIB_0821 as close as possible in structure to its natural counterpart with high and stable output, the level of purification and functional activity.

Implementation of technical tasks and the technical result is ensured by using the following essential features:

- plasmid pHisTevTSIB0821, which determines the synthesis of recombinant prolidase TSIB_0821, characterized by the presence of the following fragments: NdeI/SalI fragment of plasmid pET-22b(+) (Novagen) size 5365 P.O., NdeI/NcoI fragment encoding sntihistamine tag and website recognition/cleavage TEV protease (Seq. 1), and NcoI/SalI fragment containing the structural part of the gene TSIB_0821 corresponding to the open reading frame 749419-750516 complete genomic sequence of the archaea Thermococcus sibiricus MM 739 (gi:2423 97997), adapted by 5'- and 3'-ends of the sites restrikes Ncol and Sail (Seq. 2), connected so that when their biosynthesis in E. coli cells remained continuous reading frame;

a strain of E. coli Rosetta(DE3), transformed with plasmid pHisTevTSIB0821 according to claim 1 is inducible by producing chimeric protein comprising the amino acid sequence prolidase TSIB_0821, the slit is on the N-end with sntihistamine tag and website recognition/cleavage protease TEV;

- a method of obtaining a recombinant prolidase TSIB_0821, characterized by the fact that after addition of the inducer of the expression of the producing strains according to claim 2 incubated in liquid culture medium LB for 20 h at 18°C, and then the bacterial cells precipitated by centrifugation, the suspension of cells disintegrate in the buffer solution, the extract was centrifuged, and the supernatant was placed on a column with metalroofing resin, which, after removal of products of nonspecific binding, elute the target protein, incubated with TEV protease, which is split along its amino acid sequence is removed again metallogenetic chromatography, then the target product concentrate and the final douchemat gel filter.

The proposal is illustrated as follows.

Recombinant plasmid pHisTevTSIB0821 is a circular double-stranded DNA size 6561 BP

Recombinant E. coli strain Rosetta(DE3)/pHisTevTSIB0821 characterized by the following features:

Cultural and morphological characteristics.

Cells of strain form large round with smooth edges, convex colonies up to 5 mm in diameter, surface colonies are smooth, the consistency of mucous. The pigment does not accumulate. Gram-negative, do not form spores, capsules do not have. Colonies grow well on simple nutrient media (LB). With the growth in the Jew who their environments intensive form a smooth suspension.

Physical and biological characteristics.

The E. coli strain Rosetta(DE3)/pHisTevTSIB0821 of uidatepicker on its biochemical properties. The strain does not possess gelatinase activity, not fermented lysine, breaks down glucose, lactose, mannitol, sucrose to acid and gas. Has a mutation in the gene lac, providing control of the level of expression and broadcast six rare codons. Optimal growth temperature is 37°C, and for products TSIB_0821 - 20°C.

Antibiotic resistance

Cells of strain are characterized by resistance to chloramphenicol (34 μg/ml) and ampicillin (200 μg/ml).

Pathogenicity and toxicity

Recombinant E. coli strain Rosetta(DE3)/ pHisTevTSIB0821 not pathogenic and non-toxic to warm-blooded animals.

The strain is stored in the usual way in suspensions with glycerol (25%) at -70°C.

The inventive method of obtaining recombinant prolidase TSIB_0821 lies in the cultivation of cells of E. coli strain Rosetta(DE3)/pHisTevTSIB0821 in LB nutrient medium containing ampicillin and IPTG, the separation of the biomass from the culture fluid, the destruction of microbial cells, followed by separation of the target product from the soluble fraction of cell proteins with the following technological processes: two metalrootpaneui chromatography, gel filtration, processing TEV protease, dialysis, concentration. The yield of recombinant protein in the p is imeneniya the proposed method of isolation and purification is not less than 1 mg from 1 l of culture with the purity of more than 97%.

Recombinant prolidase TSIB_0821, the claimed isolated and purified by a method characterized by the following characteristics: a molecular weight of 41 kDa;

the optimal working conditions of the enzyme: the temperature of 85-100°C, pH 7.5-9.5, 1 mm MP2+in the incubation media; specific activity of not less than 400 IU/mg protein under the conditions of the hydrolysis at 90°C in a reaction mixture containing 50 mm MOPS, pH 8.2, 100 mm NaCI, 1 mm nl2, 5 mm peptide substrate Met-Pro and 1 µg of the enzyme. Data on physico-chemical characterization and determination of the enzymatic activity of recombinant prolidase TSIB_0821 indicate the presence in a given polypeptide, dipeptidyl-prolidase activity inherent in its natural counterpart. Recombinant prolidase TSIB_0821 can be successfully used in biotechnological processes carried out at elevated temperatures and pH, as a model for studying the molecular mechanisms for thermostability.

The advantages of the proposed method of obtaining prolidase TSIB 0821 are:

- the use of a fused gene comprising the following structural elements:

- the nucleotide sequence encoding sntihistamine tag nucleotide sequence encoding a site of recognition/cleavage protease TEV, and the nucleotide sequence of the gene TSIB_0821 connected so that when their biosyn is use in E.coli cells remained continuous reading frame, allowing after separating fused recombinant protein by the method of metalroofing chromatography to remove the additional amino acid sequence using site-specific TEV protease;

- the use of the producer strain based on the strain of E. coli Rosetta(DE3)ensuring translation of six rare codons, which allows to increase the efficiency of biosynthesis and get a full-size recombinant protein;

- use the minimum number of stages chromatographic purification, which allows to obtain pure recombinant protein in a short time with minimal loss of the protein and its functional activity.

The implementation of the invention

The way to obtain functionally active prolidase based on the use of gene TSIB_0821 thermophilic archaea Thermococcus sibiricus, fused at the N-end with a nucleotide sequence that encodes sntihistamine tag and website recognition/cleavage protease TEV, is illustrated by the following examples.

Example 1. Construction of plasmids pHisTevTSIB0821.

Recombinant plasmid pHisTevTSIB0821 containing collected in a single reading frame nucleotide sequence encoding sntihistamine , merged with site recognition/cleavage protease TEV? and the structural gene TSIB_0821, design-based commercial plasmid pET-22b(+) (Novagen), in which fra is ment NdeI/Ncol, encoding a leader peptide Rev, replaced with NdeI fragment/Ncol encoding sntihistamine tag, merged with site recognition/cleavage protease TEV.

2 μg of plasmid DNA pet-I(+) treated with restrictase Ndel and Sail (Fermentas) in the recommended buffer for 1 hour, the vector portion of the plasmid size 5365 P.O. allocate electrophoresis 1% agarose gel using the kit for DNA extraction (Fermentas) according to the manufacturer's recommendations.

The DNA fragment NdeI/Ncol encoding sntihistamine tag, merged with site recognition/cleavage protease TEV, obtained by 30 min of annealing at 75°C With subsequent cooling in ice oligonucleotides NdeHisTev For and NcoHisTev_Rev, mixed in equimolar amounts (100 pMol each):

NdeHisTev_For 5'-TATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCC

AACGACCGAAAACCTGTATTTTCAGGGCGC-3'

NcoHisTev_Rev 5'-CATGGCGCCCTGAAAATACAGGTTTTCGGTCGTTGGGATATCGTAA

TCGTGATGGTGATGGTGATGGTAGTACGACA-3'

The DNA fragment containing the full-size structural gene TSIB_0821, obtained by polymerase chain reaction using as a template the plasmid pQE80L_TSIB0821 adopted for the prototype [Trofimov A.A. et al., Acta Crystallogr Sect F Struct Biol Cryst Commun. -2012], and primers XaaP_For and XaaP_Rev where XaaP_For primer, it is specific to the 5'-end of the structural part of the gene TSIB_0821 and contains the site of restrictase Ncol, XaaP_Rev reverse primer, it is specific to the 3'-end of the structural part of the gene TSIB_0821 and provided the site restrictase Sail:

XaaPJFor: 5'- GC CC ATG GAT TAC AAG AGA AGG ATT CAT AAG -3' XaaP_Rev: 5'-TTT GTC GAC TAG HUNDRED CGT GAT CAA TTC CCT ATC-3' the reaction is carried out under the following conditions: 10x Encycio buffer, 50x mix polymerases Encycio ("Encycio PCR kit, Evrogen, Moscow), 50x mixture of 10mM dNTP, 100 ng genomic DNA of Thermococcus sibiricus MM 739. The amplification process comprises the following steps: heating at 94°C - 3 min, 30 cycles of PCR (20 - 95°C, 30 s at 58°C, 1 min 72°C) and incubation of 10 min at 72°C. the amplification Product purified by electrophoresis in 1% agarose gel with subsequent isolation from agarose using the kit for DNA extraction from gel (Fermentas). Fragment (1 μg) treated with restrictase Ncol and Sail (Fermentas) in the recommended buffer for 1 hour, and the product of the enzyme purified by electrophoresis as described above.

The resulting fragments and the vector portion of the plasmid pet-I(+) sew using a ligase reaction carried out in a volume of 20 µl with ispolzovaniem reaction buffer and enzyme T4 DNA ligase company (Fermentas) according to manufacturer's instructions. 10 μl of ligase mixture is used for transformation of competent E.coli cells Match 1. Transformants plated on LB-agar containing 125 μg/ml of ampicillin. Grown after incubation for 16 h at 37°C. single colonies Tsevaot in liquid LB medium. containing 125 μg/ml ampicillin, and grown for 16 h at 37°C in an orbital shaker at intensity swing 200 rpm Vydeleny is obtained from biomass plasmid DNA tested for the absence of mutations, incurred when cloning using automated sequencing.

Example 2. Obtaining E. coli strain Rosetta(DE3)/pHisTevTSIB0821

Producing strains obtained by transformation of cells of E. coli strain Rosetta(DES) recombinant plasmid pHisTevTSIB0821 using conventional genetic engineering techniques [Sambrook J., Fritsch E.F., Maniatis T. // Molecular Cloning. A. Laboratory Manual. 2bd ed. Cold Spring Harbor, NY, 1989]. Transformants plated on LB-agar containing 34 μg/ml chloramphenicol and 125 μg/ml of ampicillin. Grown after incubation for 16 h at 37°C. a single colony Tsevaot in 0.5 ml of liquid LB medium containing 34 μg/ml chloramphenicol and 125 μg/ml ampicillin and grown for 16 h at 37°C and then transferred into a 500 ml flask with 100 ml of liquid LB medium containing 1 g bacteriophora, 0.5 g backdragging extract and 1 g of Nad in 10 mm Tris model HC1, pH 7.0, in the presence of 125 mg/ml ampicillin, and grown on a shaker at 200 rpm and 37°C to an optical density of 0.7 (D600), then add the inducer of the expression of IPTG to a final concentration of 0.2 mm and then incubated at 18°C for 20 hours.

To determine the productivity of the strain aqueous extracts of cells analyzed by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate. Gel paint Kumasi R-250 according to standard methods and determine the relative amount of protein in the band of the target product. With the actual content of the recombinant protein is not less than 1% of all proteins in the soluble fraction.

Example 3. Isolation, purification and characterization of recombinant prolidase TSIB_0821

Night culture volume of 10 ml with seeds of the producer strain is transferred into a sterile pre-prepared fermenter RALF PLUS (Bioengineering) with 5 l of liquid LB medium containing per liter 10 g bacteriophora, 5 g backdragging extract and 10 g NaCI in 10 mm Tris model HC1, pH 7.0, in the presence of 125 mg/ml ampicillin and antifoaming reagent Antifoam 0-60 (Sigma). The cultivation is carried out in the conditions: temperature 37±0.1°C, pH 7.0±0.1, aeration of 30 l/h, the engine speed mixer 600 rpm Control the density of cells in culture is carried out using a remote sample on the spectrophotometer SmartspectPlus (BioRad) at 600 nm. Upon reaching an optical density of 0.7 (ODeoo) conduct induction by adding IPTG to 0.2 mm, followed by incubation for 20 hours at 18°C. Cells are precipitated on the Beckman centrifuge at 6000 rpm for 20 min and frozen cellular precipitation at -70°C.

Frozen biomass (about 40 g)was obtained from 5 l of bacterial culture are lysed in 100 ml of chilled buffer solution (400 mm NaCI, 50 mm HEPES pH 7.4, 5 mm imidazole)containing 0.2% Triton X-100, 5% glycerol and hibitory protease (Sigma)and treated with ultrasound using an ultrasonic disintegrator "Ultrasonic Processor" (Cole Parmer) for 5×30 s with cooling in ice. Insoluble components of the lysate precipitated what centrifugational at 20000 rpm, 20 min, 4°C. the Supernatant is collected and placed on a column with metalroofing resin Ni-NTA Superflow (Quigen), pre-equilibrated with buffer A. Metallochemistry affinity chromatography is performed using chromatographic system ACT Prime (GE Healthcare). The elution of protein is carried out with buffer a containing 500 mm imidazole. The fractions containing the target protein are pooled and incubated with TEV protease (Invitrogen) at room temperature for 2 hours, followed by dialysis against three liters of buffer solution And for 16 hours at 4°C. For exemption from castigating tag and drug TEV protease re metallochemistry chromatography. The fraction of the target protein that is not bound peroxidase resin concentrate on the centrifuge concentrator Amicon Ultra 10k (Millipore) and douchemat gel-filtration on a column of Superdex G-75 (GE Healtcare), balanced buffer solution (200 mm NaCI, 20 mm Tris-Hcl, pH 8.0, 5% glycerol, 2 mm P-mercaptoethanol and 0.1% b-octylglucoside). The yield of recombinant protein is 1 mg from 1 liter of culture at a purity of at least 97%, determined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, followed by staining of Kumasi R-250 according to standard methods. Correspondence received recombinant protein to its natural counterpart is also confirmed using Maldi-TOF-mA is with spectrometry peptide fingerprint.

Activity prolidase determined by the splitting of the various dipeptide substrates with Proline residue in the C-terminal position. The conditions of the reaction:

temperature 90°, the incubation mixture in a volume of 500 μl containing 50 mm MOPS, pH 8.2, 100 mm NaCI, pH 8.2, 1 mm nl2and 5 mm substrate, 0.1-2 μg of the enzyme. The course of the reaction is controlled spectrophotometrically at 515 nm by the formation of complex ninhydrin-Proline with extinction coefficient 4,570 M-1·cm-1what reaction is stopped by adding an equal volume of glacial acetic acid, then add 500 μl of ninhydrin reagent containing 3% ninhydrin weight./about 60% of acetic acid and 40% phosphoric acid, and incubated for 10 min at 90°C. For translating the optical units in the resultant Proline using the calibration curve. Per unit activity prolidase take the amount of enzyme that catalyzes the hydrolysis of 1 μmol of peptide substrate for 1 min Specific activity is expressed as units of enzyme activity per 1 mg of protein.

As follows from the above examples, the invention allows to obtain preparative amounts of active recombinant prolidase using relatively simple and reliable techniques for cultivating cells of the producer strain and excretion of protein.

1. Plasmid pHisTevTSIB0821 defining SinTe the recombinant prolidase TSIB_0821, characterized by the presence of the following fragments: NdeI/SalI fragment, the plasmid pET-22b(+) (Novagen) size 5365 P.O., NdeI/NcoI fragment encoding sntihistamine tag and website recognition/cleavage TEV protease (Seq. 1), and NcoI/SalI fragment containing the structural part of the gene TSIB_0821 corresponding to the open reading frame 749419-750516 complete genomic sequence of the archaea Thermococcus sibiricus MM 739 (gi:242397997), adapted by 5'- and 3'-ends of the sites restrikes Ncol and Sail (Seq. 2), connected so that when their biosynthesis in E. coli cells remained continuous reading frame.

2. The E. coli strain Rosetta(DE3), transformed with plasmid pHisTevTSIB0821 according to claim 1, - inducible production of a chimeric protein comprising the amino acid sequence prolidase TSIB_0821, fused to the N-end with sntihistamine tag and website recognition/cleavage protease TEV.

3. A method of obtaining a recombinant prolidase TSIB_0821, characterized in that after the addition of inducer of the expression of the producing strains according to claim 2 incubated in liquid culture medium LB for 20 h at 18°C, and then the bacterial cells precipitated by centrifugation, the suspension of cells disintegrate in the buffer solution, the extract was centrifuged, and the supernatant was placed on a column with metalroofing resin, which after removal of products of nonspecific binding elute the target protein, which is cuberoot with TEV protease, which, together with the split-off of its amino acid sequence is removed again metallogenetic chromatography, then the target product concentrate and the final douchemat gel-filtration.



 

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4 dwg, 2 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.

EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).

EFFECT: improvement of the method.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents the method for obtaining 2S,4R-monatin or its salt providing the contact of 4R-IHOG with aminotransferase of L-amino acid in presence of L-amino acid and method for 2S,4R-monatin or its salt obtaining with provision of 2S,4R-monatin isomerisation. The present invention also reveals aminotransferases of L-amino acid for performing the methods of monatin obtaining and method for obtaining such L-aminotransferases providing the cultivation of bacterial cell, into which there input is an expression vector containing polynucleotide that codes the presented aminotransferase.

EFFECT: invention allows obtaining 2S,4R-monatin and 2R,4R-monatin with improved output due to the use of revealed aminotransferases of L-amino acid.

27 cl, 6 dwg, 23 tbl, 48 ex

FIELD: biotechnologies.

SUBSTANCE: invention pertains to the versions of antibodies against IL-6 receptor, variable regions of light and heavy chains of which are modified by introduction of amino-acid replacement. There revealed is a pharmaceutical composition for treating the diseases associated with IL-6 and containing the said version of antibody.

EFFECT: invention allows efficient treatment of the diseases associated with IL-6.

8 cl, 48 dwg, 16 tbl, 20 ex

FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

2 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes two-stranded RNA modified with lipids and including antisense thread, the nucleotide sequence of which is complementary to target sequence in the target gene, and a sense thread, the nucleotide sequence of which is complementary to antisense thread. A two-stranded lipid is connected to the first nucleotide on the 5'-end of sense thread, either directly or through a linker. RNA has high stability to action of nuclease and is effectively absorbed with a cell, as well as generates excellent effect of RNA interference.

EFFECT: improvement of efficiency.

15 cl, 14 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention also refers to an expression vector and a transformant containing polynucleotide, as well as a production method of lipid composition or composition based on fat acids using the transformant.

EFFECT: invention allows producing a new ferment with improved properties, which has glycerol-3-phosphatocyltransferase activity.

11 cl, 4 dwg, 14 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: genetic structure pAd-SM is produced, being built by homological recombination of the vector pAdEasy-1, containing the main part of genome of adenovirus and plasmid pAdTrack-CMV, where cDNA fragments of human genes SOX2 and C-MYC are placed, being connected with a nucleotide sequence that codes P2A-peptide. The plasmid pAd-SM produced as a result of homological recombination contains a fragment SOX2-P2A-C-MYC under control of a constitutive promotor CMV.

EFFECT: possibility to produce adenovirus particles for simultaneois delivery of genes and expression of proteins SOX2 and C-MYC in human cells, pAd-SM contains a gene that codes a fluorescent protein EGFP, under control of a CMV promotor, which makes it possible to track transduction of cells and elimination of virus DNA from a cell.

3 cl, 1 dwg

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

FIELD: gene engineering, biotechnology, medicine.

SUBSTANCE: invention relates to production of polypeptides bearing specific antigen determinants of OspC protein and FlaB flagellin fragment appropriate to genus Borrelia garinii being prevalent in Russia. Recombinant plasmid pREB9-H6-F7 DNA providing synthesis of B.garnii FlaB protein fragment contains: fragment with size of 100 b.p. encoding signal amino acid sequence of Staphylococcus aureus α-toxin (CAT) and eight N-terminated amino acid residues of mature CAT; B.garnii flaB gene fragment with size of 500 b.p; nucleotide sequence in flaB gene reading frame encoding amino acid residues of glycin and 6 histidins; pIL-2/21 plasmid BamHI - EcoR1 DNA fragment with internal unique Xmal restriction site, containing β-lactamase bla gene and regulatory region of Proteus mirabilis recA gene; unique Xmal, EcoR I, BstEII and BamHI restriction sites. Recombinant plasmid pREB9-H6-C25 DNA providing synthesis of B.garnii OspC protein contains fragment with size of 660 b.p. encoding B.garnii OspC protein; nucleotide sequence in ospC gene reading frame encoding amino acid residues of glycin and 6 histidins; pIL-2/21 plasmid BamHI - EcoR1 DNA fragment with internal unique Xmal restriction site, containing β-lactamase bla gene and regulatory region of Proteus mirabilis recA gene; unique Xmal, EcoR I, BstEII and BamHI restriction sites.

EFFECT: high specific diagnosis method with improved accuracy.

2 cl, 2 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: heterologous protein is obtained by cultivation of strain Corynebacterium glutamicus AJ 12036, which does not produce cell surface protein and contains gene expressing construct wherein nucleic acid sequence encoding signal peptide of cell surface protein of Corynebacterium glutamicus or C. ammoniagenes is bound in direct direction to promoter sequence, and nucleic acid sequence encoding heterologous protein is bound in direct direction to abovementioned nucleic acid sequence encoding signal peptide. Further heterologous protein secreted from cells is isolated.

EFFECT: high effective method for heterologous protein production.

6 cl, 8 tbl, 10 ex

FIELD: biotechnology, agriculture.

SUBSTANCE: one should carry out cell transformation of hard or soft wheat varieties with, at least, two product-coding genes and blocking the nutrition of Eurygaster integryceps Puton, where, at least, one of the genes mentioned is functionally connected with a promoter that provides constitutive expression of gene's product in a plant and, at least, the second of the mentioned genes is functionally connected with a promoter that provides tissue-specific expression of gene product in a plant. Wheat plant should be regenerated out of mentioned transformed cells. The innovation suggested enables to increase resistance of wheat varieties to harmful bugs (Eurygaster integryceps Puton) and, thus, obtain high-quality grain and keep the yields.

EFFECT: higher efficiency.

4 cl, 1 dwg, 7 ex, 3 tbl

FIELD: selection and biotechnology of plants, agriculture.

SUBSTANCE: invention relates to highly effective system of genetic transformation of sugar beet plants. Method involves Agrobacterium-mediated transformation of sugar beet plant cotyledonous unit meristematic cells cultured in vitro that did not enter in the stage of callus-formation or regeneration of sprouts. Then sprouts are regenerated from transformed cells. Invention provides enhancing effectiveness and frequency of transformation of sugar beet plants.

EFFECT: improved preparing method of plants.

5 cl, 1 dwg, 2 tbl, 6 ex

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

FIELD: gene engineering, in particular production of transgenic potato with Colorado beetle resistance and biological and food safety.

SUBSTANCE: recombinant polynucleotide sequence of general formula X-A, wherein X is part of nucleotide sequence of introduced genetic construct representing in SEQ ID N 1; A is nucleotide sequence of flanking site of genomic DNA of genus Elizaveta potato, representing in SEQ ID N 2 is constructed; plant cell producing of cryIIIa protein and containing recombinant polynucleotide sequence, as well as potato transgenic plant and vegetative generation thereof with Colorado beetle resistance are obtained. Polynucleotide sequence is used in identification of plant cell producing of cryIIIa protein, plant and vegetative generation.

EFFECT: new potato genus with Colorado beetle resistance.

6 cl, 6 dwg, 7 tbl, 7 ex

FIELD: gene engineering, in particular production of transgenic potato with Colorado beetle resistance and biological and food safety.

SUBSTANCE: recombinant polynucleotide sequence of general formula X-A, wherein X is part of nucleotide sequence of introduced genetic construct representing in SEQ ID N 1; A is nucleotide sequence of flanking site of genomic DNA of genus Nevskiy potato, representing in SEQ ID N 2 is constructed; plant cell producing of cryIIIa protein and containing recombinant polynucleotide sequence, as well as potato transgenic plant and vegetative generation thereof with Colorado beetle resistance are obtained. Polynucleotide sequence is used in identification of plant cell producing of cryIIIa protein, plant and vegetative generation.

EFFECT: new potato genus with Colorado beetle resistance.

6 cl, 6 dwg, 7 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: DNA is constructed encoding protein, advancing fungus resistance to certain group of compounds. Alternatively DNA is constructed having defect in function and encoding protein, which advances lowering of GPI-anchored protein amount in fungus cell wall. Encoded protein is useful in production of antibody thereto which may by applied as active ingredient of antifungal agent.

EFFECT: new method for production of antifungal agent.

11 cl, 8 dwg, 1 tbl, 2 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for producing methanol. Method involves culturing recombinant microorganism E. coli at temperature 20-30°C. Prepared culture and cells isolated from this culture, or treated product of these cells are remained in contact with methane for producing methanol. Indicated recombinant microorganism ahs ability to convert methane to alcohol as result of transformation of DNA encoding enzyme methane oxygenase of soluble type. Invention provides expression of all components of methane oxygenase that allows realization of method for producing methanol under milder conditions.

EFFECT: improved producing method.

3 cl, 1 dwg, 1 tbl

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