RU2509154C1 - RECOMBINANT pHisTevTSIB0821 PLASMID; Escherichia coli Rosetta(DE3)/pHisTevTSIB0821 STRAIN TRANSFORMED BY ABOVE SAID PLASMID, AND METHOD FOR OBTAINING RECOMBINANT TSIB_0821 PROLIDASE - Google Patents

Abstract

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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Description

FIELD OF THE INVENTION

The present invention relates to biotechnology, in particular to genetic engineering, and relates to a method for producing a functionally active recombinant prolidase of thermostable archaea Thermococcus sibiricus (TSIB_0821), as well as a recombinant plasmid and Escherichia coli strain transformed by it to produce recombinant prolidase TSIB_0821. The invention allows the production of functionally active recombinant prolidase for use in biotechnological processes, including those carried out at elevated temperatures and pH, and also as a model for studying the molecular mechanisms of thermal stability.

State of the art

Prolidase (proline-specific dipeptidase, imidodipeptidase, E.C. 3.4.13.9., Hereinafter referred to as prolidase) is an enzyme that hydrolyzes dipeptides in which proline is in the C-terminal position (Xa-Pro or X-Pro) [Cunningham, D. & O'connor, B. 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]. Prolidases are found in eukaryotes, bacteria and archaea and are extremely promising enzymes for use in medicine and biotechnology.

In mammals, prolidases are involved in the final stages of catabolism in the cleavage of proline-rich molecules, primarily 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, prolidase deficiency causes the development of a number of connective tissue diseases [Viglio, S., Annovazzi, L., Conti, B., Genta, I., Perugini, P., Zanone, C., Casado, B., Cetta, G. & ladarola, P. The role of emerging techniques in the investigation ofprolidase deficiency: from diagnosis to, the development of a possible therapeutical approach // J Chromatogr In Analyt Technol Biomed Life Sci. 2006. -Vol. 832. -P. 1-8], for the fight against which recombinant enzymes are used as replacement therapy [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, T., 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, C., Conti, B., Perugini, P., Pavanetto, F., Modena, T., 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, increased prolidase activity is a diagnostic marker of a number of oncological diseases [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 of which they create proline-containing inactivated forms of drugs that turn into active anti-cancer drugs after cleavage by prolidases [Patent W09745117. -1997, Mittal, S, Song, X., Vig, B. 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].

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

properties and functions // FEMS Microbiol. 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 in the fermentation of cheeses and in other food industries [Courtin, P., Nardi, M., Wegmann, U, Joutsjoki, V., Ogier, JC, Gripon, J. C, Palva, A., Henrich , 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 prolidases studied today, primarily prolidases from thermophilic archaea, are active against toxic organophosphorus molecules that are part of nerve gases and pesticides [Vyas, NK, Nickitenko, A., Rastogi, V. K., Shah, SS & Quiocho, FA 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], therefore, high research activity is observed in the development of technologies aimed at the use of prolidases as biosensors for the detection of toxic substances of this class [Simonian, AL, Grimsley, J. K., Flounders, A. W, Shoeniger, JS, Cheng , T.-S., DeFrank, JJ & Wild, JR Enzyme-based biosensor for the direct detection of fluorine-containing organophosphates // Analytica Chimica Acta. -2001. -P. 15-23], biodecontamination agents [Cheng, T.C. & DeFrank, J. J. (eds.) Hydrolysis of organophosphorus compounds by bacterial prolidases // Dordrecht: Kluwer Academic Publishers. -2000] or antidote components [Petrikovics, I., Mcguinn, WD, Sylvester, D., Yuzapavik, P., Jiang, J., Way, JL, Papahadjopoulos, D., Hong, K., Yin, R., Cheng, TC & DeFrank, JJ In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism // Drug Deliv. -2000. -Vol. 7. -P. 83-89].

To date, slightly more than 10 prolidases from various organisms have been characterized, which are listed in the review [R. Semcer and A. Grunden. Prolidase function in proline metabolism and its medical and biotechnological applications // J. Appl. Microbiol. 2012. doi: 10.1111 / j.1365-2672.2012.05.053]. The prospect of the search and comprehensive study of new enzymes of this class, especially from extremophilic archaea, as well as the cloning of the corresponding genes and the production of recombinant proteases, including those with directionally modified methods of genetic engineering properties, is confirmed by publications both in periodical scientific journals and in 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 proline-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 Microbiol 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 Microbiol 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 work demonstrates that in order to make fuller use of the biotechnological potential of prolidases, two strategies must be combined: first, to study new enzymes, and secondly, to optimize the properties of natural enzymes in accordance with the requirements of the technological process. Of particular interest in this regard are prolidases from extremophilic microorganisms, since they have a wide range of unique properties, namely they are able to work in extreme conditions: elevated temperature, acidic or alkaline pH, high ionic strength, the presence of high concentrations of detergents. Therefore, to search for genes encoding new prolidases, the gene of the hyperthermophilic archaea Thermococcus Sibiricus MM739, which lives in hydrothermal springs and underground oil reservoirs in Western Siberia at temperatures from 40 ° C to 88 ° C, was selected. Analysis of the decrypted T. Sibiricus genome reveals the presence of the TSIB 0821 gene (GenBank accession number ACS89882.1), which encodes a protein similar to the previously studied thermophilic protease from Pyrococcus horikoshii OT3 (52% homology).

As a prototype, the method of obtaining prolidase TSIB_0821 fused at the N-terminus with the sequence MRGSHHHHHHGS [Trofimov, AA, Slutskaya, E. A., Polyakov, K. M., Dorovatovskiic, PV, Gumerov, V. M., Popov, V O. Influence of intermolecular contacts on the structure of recombinant prolidase from Thermococcus sibiricus // Acta Crystallogr Sect F Struct Biol Cryst Commun. -2012]. This method involves amplification of the TSIB_0821 gene (GenBank accession number ACS89882.1) on the T. Sibiricus genomic DNA matrix using a polymerase chain reaction, its cloning into the pQE80L expression vector (Qiagen), and the preparation of Escherichia coli Rosetta-gami strain (DE3) ( Novagen), transformed with plasmid pQE80L_TSIB0821, cell growth of the producer strain, induction of expression and isolation of the recombinant protein from the resulting biomass. The disadvantage of this method is the inability to remove the amino acid sequence of the six-histidine tag from the N-terminus of the recombinant prolidase TSIB_0821, which, according to the X-ray diffraction analysis of the fusion protein MRGSHHHHHHGS-TSIB_0821, is actively involved in the formation of the spatial folding of the polypeptide, negatively affecting its functional activity and resistance to exposure, which makes the recombinant prolidase TSIB_0821, obtained by this method, unsuitable for use in biotechnology biological processes.

Disclosure of invention

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

The problem is solved by the proposed method, which involves the construction of a plasmid pHisTevTSIB0821 encoding the synthesis of recombinant prolidase TSIB_0821, fused at the N-terminus with a six-histidine tag tag and a TEV protease recognition / cleavage site, and the E. coli Rosetta strain (DE3IB) strain DE21IB / DE21 derived from this plasmid a producer of recombinant prolidase TSIB_0821 in the form of a fusion protein and the development of a method for the isolation and purification of recombinant prolidase TSIB_0821, which ensures the separation of the N-terminal amino acid sequence tigistidinovogo tag from the target polypeptide.

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

The implementation of the technical task and obtaining the technical result is ensured by the use of the following set of essential features:

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

- E. coli Rosetta strain (DE3), transformed with the plasmid pHisTevTSIB0821 according to claim 1, is an inducible producer of a chimeric protein, including the amino acid sequence of prolifease TSIB_0821, fused at the N-terminus with a six-histidine tag and a TEV protease recognition / cleavage site;

- a method of producing a recombinant prolidase TSIB_0821, characterized in that after the addition of an expression inducer, the producer strain according to claim 2 is incubated in LB liquid medium for 20 hours at 18 ° C, then the bacterial cells are precipitated by centrifugation, the cell suspension is disintegrated in a buffer solution, the extract is centrifuged, and the supernatant is placed on a metal affinity resin column, with which, after removal of the products of non-specific binding, the target protein is eluted, which is incubated with the TEV protease, which cleaved its amino acid sequence removed retransmission metal chelate chromatography, then concentrated and the desired product finally doochischayut gel filtration.

The proposal is explained as follows.

Recombinant plasmid pHisTevTSIB0821 is a 6561 bp double-stranded circular DNA.

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

Cultural and morphological characters.

The cells of the strain form large round with smooth edges, convex colonies up to 5 mm in diameter, the surface of the colonies is smooth, the mucous consistency. The pigment does not accumulate. Gram-negative, do not form a spore, do not have capsules. Colonies grow well on simple nutrient media (LB). When growing in liquid media, they form an intense smooth haze.

Physico-biological signs.

The strain E. coli Rosetta (DE3) / pHisTevTSIB0821 is speciesotypical in its biochemical properties. The strain does not have gelatinase activity, does not ferment lysine, breaks down glucose, lactose, mannitol, sucrose to acid and gas. It has a mutation in the lac gene, which provides control of the expression level, as well as translation of six rare codons. The optimum temperature for growth is 37 ° C, and for production TSIB_0821 - 20 ° C.

Antibiotic resistance

The cells of the strain are resistant to chloramphenicol (34 μg / ml) and ampicillin (up to 200 μg / ml).

Pathogenicity and toxicity

The recombinant E. coli Rosetta (DE3) / pHisTevTSIB0821 strain is not pathogenic and toxic to warm-blooded animals.

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

The inventive method for producing recombinant prolidase TSIB_0821 consists in culturing E. coli cells of strain Rosetta (DE3) / pHisTevTSIB0821 in LB nutrient medium containing ampicillin and IPTG, separating biomass from the culture fluid, destroying microbial cells, and then isolating the target product from the soluble cell protein fraction with using the following technological processes: two metal affinity chromatography, gel filtration, TEV protease treatment, dialysis, concentration. The output of the recombinant protein as a result of the application of the proposed method of isolation and purification is at least 1 mg from 1 l of culture with a purity of more than 97%.

Recombinant prolidase TSIB_0821, isolated and purified by the claimed method, is characterized by the following features: molecular weight 41 kDa;

optimal conditions of the enzyme: temperature 85-100 ° C, pH 7.5-9.5, 1 mm Mn ²⁺ in an incubation medium; specific activity of at least 400 u / mg protein, provided that hydrolysis is carried out at 90 ° C in a reaction mixture containing 50 mM MOPS, pH 8.2, 100 mM NaCI, 1 mM MnCl ₂ , 5 mM Met-Pro peptide substrate and 1 μg of enzyme . The data on the physicochemical characterization and determination of the enzymatic activity of the recombinant prolidase TSIB_0821 indicate the presence of the dipeptidyl prolidase activity inherent to its natural analogue in the studied polypeptide. Recombinant prolidase TSIB_0821 can be successfully used in biotechnological processes carried out at elevated temperatures and pH, as well as as a model for studying the molecular mechanisms of thermal stability.

The advantages of the proposed method for producing prolidase TSIB 0821 are:

- the use of a fused gene, consisting of the following structural elements:

- a nucleotide sequence encoding a six-histidine tag, a nucleotide sequence encoding a TEV protease recognition / cleavage site, and a nucleotide sequence of the TSIB_0821 gene, connected so that during their biosynthesis in E. coli cells a continuous reading frame is maintained, which allows, after isolation of the fused recombinant protein, the method metal affinity chromatography to remove additional amino acid sequences using site-specific TEV protease;

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

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

The implementation of the invention

A method for producing a functionally active prolidase based on the use of the thermophilic archaea gene Thermococcus sibiricus TSIB_0821 fused at the N-terminus with a nucleotide sequence encoding a six-histidine tag and a TEV protease recognition / cleavage site is illustrated by the following examples.

Example 1. Construction of the plasmid pHisTevTSIB0821.

Recombinant plasmid pHisTevTSIB0821, containing assembled into a single reading frame nucleotide sequences encoding a six-histidine fused to the TEV protease recognition / cleavage site? and the TSIB_0821 structural gene, are constructed on the basis of the commercial plasmid pET-22b (+) (Novagen), in which the NdeI / Ncol fragment encoding the pe1B leader peptide is replaced by the NdeI / Ncol fragment encoding a six-histidine tag fused to the protease recognition / cleavage site TEV.

2 μg of plasmid DNA pET-22b (+) is treated with restriction enzymes Ndel and Sail (Fermentas) in the recommended buffer for 1 hour, the vector part of the plasmid size 5365 bp Electrophoretically isolated from 1% agarose gel using a DNA isolation kit (Fermentas) in accordance with the manufacturer's recommendations.

An NdeI / Ncol DNA fragment encoding a six-histidine tag fused to the TEV protease recognition / cleavage site is obtained by annealing for 30 minutes at 75 ° C followed by cooling in ice of NdeHisTev For and NcoHisTev_Rev oligonucleotides mixed in equimolar amounts (100 pMol each)

NdeHisTev_For 5'-TATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCC

AACGACCGAAAACCTGTATTTTCAGGGCCC-3 '

NcoHisTev_Rev 5'-CATGGCGCCCTGAAAATACAGGTTTTCGGTCGTTGGGATATCGTAA

TCGTGATGGTGATGGTGATGGTAGTACGACA-3 '

A DNA fragment containing the full-sized structural gene TSIB_0821 is obtained by polymerase chain reaction using the plasmid pQE80L_TSIB0821 adopted as 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 is a primer specific for the 5'-end of the structural part of the TSIB_0821 gene and contains the Ncol restriction site, XaaP_Rev is a reverse primer specific for the 3'-end of the structural part of the TSIB_08 gene and containing the restriction site Sail:

XaaPJFor: 5'-GC CC ATG GAT TAC AAG AGA AGG ATT CAT AAG-3 'XaaP_Rev: 5'-TTT GTC GAC STA TAG CGT GAT CAA TTC CCT ATC-3' This reaction is carried out under the following conditions: 10x Encycio buffer, 50x Encycio polymerase mixture (Encycio PCR kit, Eurogen, Moscow), 50x 10mM dNTP mixture, 100 ng of Thermococcus sibiricus MM 739 genomic DNA. The amplification process consists of the following stages: heating at 94 ° C for 3 min, 30 PCR cycles (20 s - 95 ° С, 30 s - 58 ° С, 1 min - 72 ° С) and incubation for 10 min at 72 ° С. The amplification product is purified electrophoretically in a 1% agarose gel, followed by isolation from agarose using a gel isolation kit (Fermentas). The fragment (1 μg) was treated with restriction enzymes Ncol and Sail (Fermentas) in the recommended buffer for 1 hour, and the restriction product was purified electrophoretically as described above.

The obtained fragments and the vector part of the plasmid pET-22b (+) are crosslinked using a ligase reaction carried out in a volume of 20 μl using the reaction buffer and T4 enzyme DNA ligase (Fermentas) according to the manufacturer's instructions. 10 μl of the ligase mixture is used to transform competent E. coli Match 1 cells. Transformants are seeded on LB agar containing 125 μg / ml ampicillin. Single colonies grown after incubation for 16 hours at 37 ° C are seeded into LB liquid medium. containing 125 μg / ml ampicillin and grown for 16 hours at 37 ° C in an orbital rocking chair at a rocking intensity of 200 rpm. The plasmid DNA isolated from the obtained biomass is checked for the absence of mutations that occurred during cloning using automatic sequencing.

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

The producer strain is obtained by transforming E. coli Rosetta strain (DES) cells with the recombinant plasmid pHisTevTSIB0821 using traditional genetic engineering technology [Sambrook J., Fritsch EF, Maniatis T. // Molecular Cloning. A. Laboratory Manual. 2bd ed. Cold Spring Harbor, NY 1989]. Transformants are plated on LB agar containing 34 μg / ml chloramphenicol and 125 μg / ml ampicillin. A single colony grown after incubation for 16 hours at 37 ° C is sown in 0.5 ml of LB liquid medium containing 34 μg / ml of chloramphenicol and 125 μg / ml of ampicillin and grown for 16 hours at 37 ° C, and then transferred to 500 ml a flask with 100 ml of LB liquid medium containing 1 g of bactotryptone, 0.5 g of bacterium yeast extract and 1 g of Nad in 10 mm Tris HCl pH 7.0, in the presence of 125 mg / ml ampicillin, and grown on a shaker at 200 rpm and a temperature of 37 ° C to an optical density of 0.7 (OD ₆₀₀ ), then an IPTG expression inducer is added to a final concentration of 0.2 mM and incubated further at 18 ° C for 20 hours.

To determine the productivity of the strain, the aqueous extracts of the cells are analyzed by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate. The gel is stained with Coomassie R-250 by a standard method and the relative amount of protein in the strip of the target product is determined. The recombinant protein content is at least 1% of all proteins of the soluble fraction.

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

A 10 ml overnight culture with seed of the producer strain was sterile transferred to a pre-prepared RALF PLUS fermenter (Bioengineering) with 5 L of LB liquid medium containing 10 g of bactotryptone, 5 g of bacterium yeast extract and 10 g of NaCI in 10 mm Tris HCl, pH 10 7.0, in the presence of 125 mg / ml ampicillin and Antifoam 0-60 antifoam reagent (Sigma). Cultivation is carried out under conditions: medium temperature 37 ± 0.1 ° C, pH 7.0 ± 0.1, aeration 30 l / h, stirrer speed 600 rpm. Cell density control in the culture is carried out by the method of remote sampling on a SmartspectPlus spectrophotometer (BioRad) at 600 nm. Upon reaching an optical density of 0.7 (ODeoo), induction is carried out by adding IPTG to 0.2 mM, followed by incubation for 20 hours at 18 ° C. Cells are pelleted in a Beckman centrifuge at 6,000 rpm for 20 minutes and cell pellets are frozen at -70 ° C.

Frozen biomass (about 40 g) obtained from 5 l of the bacterial culture is lysed in 100 ml of chilled buffer solution A (400 mM NaCI, 50 mM Hepes pH 7.4, 5 mM imidazole), also containing 0.2% Triton X-100, 5% glycerol and protease inhibitors (Sigma), and sonicated using an ultrasonic disintegrator "Ultrasonic Processor" (Cole Parmer) for 5 × 30 s, cooling in ice. Insoluble components of the lysate precipitated by centrifugation at 20,000 rpm, 20 min, 4 ° C. The supernatant was collected and placed on a Ni-NTA Superflow (Quigen) metal affinity resin column, previously equilibrated with buffer A. Metal chelate affinity chromatography was performed using an AKTA Prime chromatographic system (GE Healthcare). Protein elution is carried out with buffer solution A containing 500 mM imidazole. The fractions containing the target protein are combined and incubated with TEV protease (Invitrogen) at room temperature for 2 hours, followed by dialysis against three liters of buffer solution A for 16 hours at 4 ° C. To liberate from the six-histidine tag and the TEV protease preparation, repeated metal chelate chromatography is performed. The non-resin bound target protein fraction was concentrated on an Amicon Ultra 10k centrifuge concentrator (Millipore) and further purified by gel filtration on a Superdex G-75 column (GE Healtcare), equilibrated with buffer solution (200 mM NaCI, 20 mM Tris-Hcl, pH 8.0, 5% glycerol, 2 mM P-mercaptoethanol and 0.1% b-octylglucopyranoside). The recombinant protein yield is 1 mg per 1 liter of culture with a purity of at least 97%, determined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, followed by Coomassie R-250 staining according to standard methods. The correspondence of the obtained recombinant protein to its natural analogue is also confirmed by peptide fingerprint Maldi-TOF mass spectrometry.

Prolidase activity is determined by cleavage of various dipeptide substrates with a proline residue at the C-terminal position. Reaction conditions:

temperature 90 °, the incubation mixture in a volume of 500 μl contains 50 mm MOPS, pH 8.2, 100 mm NaCl, pH 8.2, 1 mm Mn ₂ and 5 mm substrate, 0.1-2 μg of the enzyme. The progress of the reaction is monitored spectrophotometrically at 515 nm to form a ninhydrin-proline complex with an extinction coefficient of 4.570 M ^-1 · cm ^-1 , for which the reaction is stopped by adding an equal volume of glacial acetic acid, then 500 μl of ninhydrin reagent containing 3% ninhydrin weight./ about, 60% acetic acid and 40% phosphoric acid, and incubated for 10 min at 90 ° C. To convert optical units to the amount of proline formed, a calibration curve is used. The unit of prolidase activity is the amount of enzyme that catalyzes the hydrolysis of 1 μmol of peptide substrate in 1 min The specific activity is expressed in units of enzyme activity per 1 mg of protein.

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

Claims (3)

1. The plasmid pHisTevTSIB0821, which determines the synthesis of recombinant prolidase TSIB_0821, characterized by the presence of the following fragments: NdeI / SalI fragment, plasmid pET-22b (+) (Novagen) 5365 bp, NdeI / NcoI fragment encoding a six-histidine tag and recognition / cleavage of the TEV protease (Seq. 1), and the NcoI / SalI fragment containing the structural part of the TSIB_0821 gene corresponding to the open reading frame 749419-750516 of the complete genomic sequence of the archaea Thermococcus sibiricus MM 739 (gi: 242397997), adapted according to 5'- and 3'-ends of the restriction sites Ncol and Sail (Seq. 2), connected so that during their biosynthesis se in E.coli cells maintained a continuous reading frame. 2. The strain E. coli Rosetta (DE3), transformed with the plasmid pHisTevTSIB0821 according to claim 1, is an inducible producer of a chimeric protein comprising the amino acid sequence of prolidase TSIB_0821 fused at the N-terminus with a six-histidine tag and a TEV protease recognition / cleavage site. 3. The method of producing recombinant prolidase TSIB_0821, characterized in that after adding the expression inducer, the producer strain according to claim 2 is incubated in LB liquid medium for 20 hours at 18 ° C, then the bacterial cells are precipitated by centrifugation, the cell suspension is disintegrated in a buffer solution , the extract is centrifuged, and the supernatant is placed on a metal affinity resin column, with which, after removal of nonspecific binding products, the target protein is eluted, which is incubated with TEV protease, which is mixed with those with the amino acid sequence cleaved by it are removed by repeated metal chelate chromatography, then the target product is concentrated and finally purified by gel filtration.