RU2564120C2 - STRAIN OF Escherichia coli BACTERIA - PRODUCENT OF HEAT SHOCK PROTEIN 70 AND METHOD FOR OBTAINING PREPARATION OF HUMAN HEAT SHOCK PROTEIN - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with a strain of bacteria Escherichia coli - a producent of a heat shock protein (HSP70) and a method of the said protein obtaining. The characterised strain is deposited on 28.11.2012 in the Russian National Collection of Industrial Microorganisms (VKPM) of FSUE State Research Institute of Genetics and Selection of Industrial Microorganisms under No B-11388. The claimed method includes the cultivation of a recombinant strain-producent, separation of the recombinant protein from the bacterial biomass, its purification from admixtures of bacterial proteins with the application of Escherichia coli strain BB 1553, transformed by vector plasmid pMSHsp70, as the strain-producent. Separation of the protein is carried out by the repeated freezing-defrosting of cells and processing the obtained suspension successively with a DNAse solution and magnesium chloride solution with the further centrifugation of the protein-containing solution and chromatographic purification of the supernatant.

EFFECT: claimed inventions make it possible to obtain the protein HSP70 with high output and high purity with the further possibility of using it for obtaining a medication.

5 cl, 1 dwg, 1 ex

Description

The invention relates to biotechnology, in particular to a technology for producing heat shock protein using genetic engineering methods.

Heat shock proteins (HSPs, HSP) belong to the family of highly conserved intracellular proteins, which play an important role in ensuring homeostasis of the body, including under extreme conditions. HSPs are divided into several families depending on their molecular weight: HSPH (Hsp110), HSPC (Hsp90), HSPA (Hsp70), DNAJ (Hsp40), the family of small HSPB (sHSP), and the family of chaperonins HSPD / E (HSP60 / HSP10 ) (Kampinga HH, Hageman J., Vos MJ, et al. Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones. 2009; 14 (1): 105-111). The proteins of the HSP70 family (Feder ME, Hofmann GE Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology // Annu. Rev. Physiol. - 1999. - Vol. 61. - P .243-282; H. Hauser, L. Shen, QL Gu et al. Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines // Gene Ther. - 2004. - Vol.11, No. 11. - P.924-932.).

Proteins belonging to the HSP70 family are induced in the cells and tissues of all known organisms, including humans, under the influence of a huge number of factors of abiogenic and natural origin. To date, the protective effect of HSP70 has been studied in sufficient detail when exposed to various pathogenetic factors, including salts of heavy metals, cytotoxic drugs, various chemical compounds, which are adverse concomitant factors of chemical production, which makes them promising for the creation of related anticancer drugs in particular, with the treatment of pathologies caused by the human papilloma virus (warts, cancer of the cervix and rectum, etc.) (RU 2282461 , 2006).

Natural protein preparations of mammalian HSP70 were isolated from tumor tissues (P.K. Sriva stava. Purification of heat shock protein-peptide complexes for use in vaccination against cancers and intracellular pathogens. Methods, 1997 Jun, 12 (2): 165-71). These methods included the accumulation and homogenization of tissue, the extraction of the target protein, as well as the fractionation of the drug using several chromatographic steps. The main disadvantages of obtaining HSP from natural sources are the need for the accumulation of tumor tissue, the complexity of the isolation process, and a small final yield.

Genetic engineering methods allow you to produce preparative amounts of HSP necessary for the study of various functions of the protein. To obtain HSP70 by genetic engineering technology, it was proposed, in particular, to use the M. tuberculosis strain containing recombinant plasmid DNA (pE HSP70), which is characterized by the fact that it is a pQE30 vector having a size of 3461 nucleotide base pairs with a lac promoter , the ampicillin resistance gene and a sequence of 18 nucleotides encoding 6 histidine residues into which the ESAT6 protein gene is operatively inserted having 288 nucleotides (between the restriction enzyme sites BglII and BamHI) and the heat shock protein gene HSP70 M. tuberculosis, I have total 1878 nucleotides (between restriction enzyme sites BamHI and HindIII) (RU 2262351, 2005). The protein thus obtained was found to be promising as a component of a vaccine for the treatment and prevention of tuberculosis infection containing a mixture of the hybrid recombinant protein ESAT-HSP70 M. tuberculosis and the protein HSP65 M. tuberculosis, an inert carrier and thymogen. However, information on the anticancer effect of the protein thus obtained was not found in the literature.

A known technology, including recombinant DNA and a producer strain of E. coli, which is capable of producing recombinant mycobacterial HSP70 in the form of a conjugate to the human immunodeficiency virus p24 protein. According to the known method, HSP70 expression is carried out in the form of a chimeric protein with p24 (Suzue K.J. and Young R.A. J. Immunol., 156: 873-879, 1996).

However, the production of a chimeric protein takes place with a small yield using complex technology, which does not allow one to consider the prospects of using it as a component of drugs or vaccines.

Known for the production of HSP70 in E. coli cells containing the pET-11 vector (Studier, FW, et al. Methods Enzymol. 185: 60-89), followed by purification of the target product using anion exchange chromatography on DEAE-Sepharose and ATP agarose affinity chromatography (K. ABRAVAYA et all., Genes Dev., 1992, V.6, p. 1153-1164), followed by concentration by ultrafiltration and desalination on glutathione-sepharose.

The disadvantage of this method is the multistage technology for the preparation and purification of HSP70, which leads to large losses of the target product. Unfortunately, there are no data on the final yield and purity of HSP70.

The recombinant human HSP70 biosynthesis corresponding to the HSPA1L gene was carried out in cells of the E. coli strain BL21 (DE3) (S. Jindal, P. Murrey, S. Rosenberg, RA Young and KP Williams. Human stress protein hsp70: overexpression in E. coli , purification and characterization. Biotechnology, 1995, 13: 1105-9). For this, HSP70 cDNA was inserted at the NdeI-BamHI restriction sites into the expression vector pET-3a. After transformation of the E. coli strain BL21 (DE3) with the obtained recombinant plasmid and induction of isopropylthiogalactoside gene expression, the target protein accumulated in the cells as inclusion bodies. Isolation of the pre-refolded HSP70 protein was based on affinity chromatography using ATP agarose.

The disadvantages of this method include the contamination of the target DnaK protein, a bacterial analogue of the eukaryotic HSP70 and, as a result, the yield of the target product is not high enough.

The closest in technical essence is the method of obtaining another representative of the HSP70 family, encoded by the HSPA1B gene (Gene Bank NM 005346) of the heat shock protein preparation, by expressing the gene of such a protein in E. coli cells, followed by isolation, refolding and purification (RU 2333956, 2008) . In this case, gene expression is carried out as part of a vector cDNA containing the T7 promoter, kanamycin resistance gene, replicative origin pUC ori and the gene encoding the lac inducer, vector DNA is cultured as a part of E. coli BL21 cells in a nutrient medium with the addition of kanamycin (100 μg / ml) and glucose (0.2%) to a turbidity level of 0.5-0.6 OD600, 0.2 mM isopropyl beta-D-thiogalactoside (IPTG) was added to the medium and continued to cultivate for 1-3 hours until the final product accumulated. Obtaining recombinant hHSP70A1B from producer cells involves several steps. Bacteria are separated from the culture medium and destroyed by one of the commonly used methods. By centrifugation, the soluble cell fraction is separated from the inclusion bodies. The latter are washed with a buffer solution from the water-soluble components of the cell, solubilization, restoration and refolding of the recombinant protein are carried out. The final purification of the target protein is carried out by metal chelate chromatography. The yield of recombinant hHSP70A1B obtained in this way is about 10 mg per 1 liter of bacterial culture. Isolation of recombinant hHSP70A1B from a soluble cell fraction is carried out in one stage - by the method of metal chelate chromatography. The yield of the target protein is approximately 30 mg per 1 liter of bacterial culture.

The disadvantage of this method is the insufficiently high yield of the target product, as well as the insufficient antitumor effectiveness of the obtained product.

The technical problem solved by the authors was the creation of a more efficient way to obtain and allocate HSP70.

The technical task was solved as a result of the creation of the E. coli strain BB1553 pMSHsp70 deposited in the All-Russian collection of industrial microorganisms (VKPM) on November 28, 2012 under the number B-11388, and the technology for the isolation of HSP70, which provides an increased yield of HSP70.

The technical result was achieved by the creation of the producer strain Escherichia coli BB 1553 developed by the authors and transformed with the vector plasmid pMSHsp70, and the preparation of HSP70 by cultivation of the E. coli strain BB1553 pMSHsp70 followed by destruction of E. coli cells by repeated freezing-expansion and treatment of the resulting suspension with a DNA solution zy and a solution of magnesium chloride, obtaining a protein-containing solution by centrifugation and protein purification by chromatographic purification on an ion-exchange sorbent DEAE-Sepharose equilibrium buffer solution at pH 7.5 and affinity chromatography on ATP agarose, after which the solution obtained after affinity chromatography was purified from lipopolysaccharides by incubation with Polymyxin B-Agarose gel and the protein was separated from the sorbent by centrifugation.

Recipient strain of E. coli BB1553 obtained at the Massachusetts Institute of Technology (USA). Physiological and biochemical characteristics of the strain are typical for Escherichia coli. Catalopositive. Oxidase-negative. Optional anaerobes. D-glucose, L-arabinose, D-xylose, sucrose, lactose, maltose, mannitol, D-mannose, L-rhamnose, sorbitol, trigallose are catabolized with the formation of acid (and gas). Do not hydrolyze gelatin. Urease (-), indole (+), tryptophandesaminase (-), lysine decarboxylase (+), growth in the presence of KCN (-), malonate is not used, the pigment is not formed. The reaction of the Voges-Proskauer is negative. They do not form H ₂ S. Do not grow on Simmons citrate medium.

The recombinant plasmid pMS-HSP70 was previously used to obtain mutant proteins (V.F. LAZAREV, “MECHANISMS OF AGGREGATION OF MUTANT BEIGS IN THE MODELS OF THE HUNTINGTON AND AMYOTROPHIC LATERAL SCLEROSIS”, abstract of abstracts, 2013, Abstract 2013_05_31_lazarev_autoref.pdf; diss, seluk.ru / / / 675697-1-mehanizmi-agregacii-mutantnih-belkov-model ...).

The plasmid was introduced into the recipient strain of E. coli BB1553 using the standard method of Ca ²⁺ -dependent cell transformation followed by selection of the target transformants on an agar medium containing ampicillin (Amp) at a concentration of 50 μg / ml.

The strain of E. coli BB1553 pMSHsp70 is characterized by the following cultural-morphological and physico-biochemical properties:

Cultural and morphological features of the strain GIMM . Gram-negative rods of oval shape with rounded edges, size 2.0-3.0 × l.5-5.0 microns, often single, but there are chains of cells of 2-4 pieces, spores and capsules do not form. The colonies on nutrient agar LB are smooth, slightly convex, with a smooth edge. In liquid media form a uniform turbidity.

The method, conditions and composition of the media for long-term storage of the strain GIMM. The strain is stored lyophilized (up to 2 years), cryopreserved at -70 ° C (up to 6 months) in 15% glycerol solution (or 7% dimethyl sulfoxide solution) in LB nutrient medium (Bactotripton - 10 g / l, yeast extract - 5 g / l, sodium chloride - 10 g / l, pH 6.8).

The method, conditions and composition of the media for the propagation of the strain GIMM. The strain is grown at 37 ° C in LB broth containing ampicillin and chloramphenicol each at a concentration of 100 μg / cm ³ . Protein isolation and purification was carried out by three-stage chromatography. In the first stage, the supernatant containing HSP70 was applied to a DEAE-Sepharose sorbent column. The column was pre-equilibrated with five volumes of a buffer solution containing 20 mM Tris-HCl, 20 mM NaCl, 0.1 mM EDTA at pH 7.5 (buffer A / 2), and two volumes of the same buffer solution containing 0.1% Triton X-100 and 0.2 mM phenyl methyl sulfonyl fluoride (PMSF). After sorption, the column was washed with two volumes of buffer solution of 40 mM Tris-HCl, 40 mM NaCl, 0.2 mM EDTA at pH 7.5 (buffer A) with 0.1% Triton X-100. Elution was performed with 1.5 volumes of 0.35 M NaCl in buffer.

In the second stage, affinity chromatography on a column with ATP agarose was used. Before the second stage, the eluate with DEAE of Sepharose was diluted 5 times with a buffer containing 20 mM Tris-HCl, 1 mM MgCl ₂ at pH 7.5 (buffer B). The resulting solution was applied to a column of ATP-Sepharose, balanced with 20 volumes of buffer B. Then the column was washed with 20 volumes of buffer B; 10 volumes of 1 mM NaCl in buffer solution B and 20 volumes of buffer B. Elution was performed with 7 volumes of 3 mm adenosine triphosphate (ATP) dissolved in the same buffer A.

In the third stage, lipopolysaccharides (LPS) were removed from the purified HSP70 solution. For this purpose, the purified protein solution obtained after affinity chromatography was incubated with Polymyxin B-Agarose gel manufactured by Pierce Biotechnology in a ratio of 100 mg HSP70 - 10 ml gel.

The concentration of protein in the eluate was determined by the method of Bradford. The yield of HSP70 protein with 1 g of biomass was 2 mg. Protein homogeneity was evaluated by SDS page electrophoresis. Protein purity was 99%.

The dosage form of a new drug in the form of a solution or lyophilisate for the preparation of an injection solution of 0.5 mg of protein in an ampoule showed high efficiency in the treatment of neoplasms.

The invention is illustrated by the following specific examples of the use of strain E. coli BB1553.

EXAMPLE 1 To cultivate the strain E. coli BB1553 pMS-HSP70 on solid medium on Petri dishes in a dry-air thermostat, standard agarized medium LB medium with the addition of two antibiotics was used, one of which was ampicillin at a concentration of 100 mg / l, and the second was streptomycin or chloramphenicol at a concentration 100 or 25 mg / l, respectively.

The strain was fermented at 37 ° C in complete LB nutrient medium (240 g of casein acid hydrolyzate, 120 g of yeast extract, 18.85 g of Na ₂ HPO ₄ * 12H ₂ O, 3.75 g of KH ₂ PO ₄ , 300 ml of 60% glucose solution, 24 ml of 1M MgSO ₄ , 10 ml of CaCl ₂ , the volume was adjusted with sterile distilled water to 1 l), pH 6.8, with the addition of the antibiotic ampicillin at a concentration of 100 mg / l. For this, several colonies from a Petri dish were seeded and a night culture of E. Coli strain was grown on a rocking chair at 37 ° C in complete LB medium with ampicillin (100 mg / L) and streptomycin or chloramphenicol (100 or 25 mg / L, respectively). After 12 hours, the culture was diluted 1: 100 with fresh LB medium with ampicillin (100 mg / L) and cultivation was continued to OD600 = (0.8.1.0). After that, 1,2-isopropyl-thio-beta-galactoside (TIN) was added to the culture to a concentration of 0.5 mM. 4-5 hours after the addition of IPTG, the cell mass was concentrated by centrifugation in a flow centrifuge at 17000 g. Then the cells were frozen at a temperature of -70 ° C.

To obtain HSP, cell biomass weighing 140 g was thawed at room temperature for 2 hours. After that, 100 ml of buffer containing 10 mM Tris-HCl and 20 mM NaCl, pH 7.6 and lysozyme to a final concentration of 0.1 mg / ml was added to it. Immediately after this, the suspension was intensively mixed in a blender for 2 minutes and then incubated with constant stirring in a beaker on a magnetic stirrer for 30 minutes at room temperature, transferred to a container and frozen at -70 ° C for 4 hours. Then the suspension was thawed in the refrigerator at + 4 ° C for 15-16 hours. The freezing and thawing procedure was repeated twice.

After repeated thawing, 60 μl of the mother solution of DNA-Zy (C = 25 mg / ml) and a solution of magnesium chloride to a final concentration of 10 mM were added to the suspension, intensively mixed on a blender for 2 min, after which its volume was brought to 400 ml by adding the same buffer solution and incubated for 90 min on a magnetic stirrer at 37 ° C.

After incubation, the suspension was centrifuged at 13 thousand rpm for 30 minutes, the supernatant after centrifugation was taken into a container (sample 1), and the precipitate was suspended in 200 ml with a blender, 200 ml of the same buffer containing 10 mM EDTA was added to the suspension, was stirred on a magnetic stirrer at room temperature for 30 minutes and again centrifuged at 13 thousand rpm for 30 minutes. The supernatant after centrifugation was taken into a container (sample 2), and the precipitate was removed. Supernatants (samples 1 and 2) were combined, centrifuged for 30 minutes at 19 thousand rpm and subjected to a three-stage chromatographic purification.

In the first stage, the combined supernatant was applied to a column with a 200 ml DEAE-Sepharose ion-exchange sorbent. The column was pre-balanced with five volumes containing 20 mM Tris-HCl, 20 mM NaCl, 0.1 mM EDTA at pH 7.5 (buffer A / 2), and two volumes of the same buffer solution containing 0.1% Triton X- 100 and 0.2 mM phenyl methyl sulfonyl fluoride (PMSF). After sorption, the column was washed with two volumes of buffer solution of 40 mM Tris-HCl, 40 mM NaCl, 0.2 mM EDTA at pH 7.5 (buffer A) with 0.1% Triton X-100. Elution was performed with 1.5 volumes of 0.35 M NaCl in buffer.

In the second stage, affinity chromatography on a column with ATP agarose (Sigma, USA) was used. Before the second stage, the eluate with DEAE-Sepharose was diluted 5 times with buffer containing 20 mm Tris-HCl, 1 mm MgCl ₂ at pH 7.5 (buffer B). The resulting solution was applied to a 1 ml ATP-Sepharose column, equilibrated with 20 volumes of buffer B. Then the column was washed with 20 volumes of buffer B; 10 volumes of 1 mM NaCl in buffer solution B and 20 volumes of buffer B. Elution was performed with 7 volumes of 3 mm adenosine triphosphate (ATP) dissolved in the same buffer A.

The concentration of protein in the eluate was determined by the method of Bradford. The yield of HSP70 protein was 280 mg. Protein homogeneity was evaluated by SDS page electrophoresis. Protein purity was 99%.

The biological activity of the obtained protein was evaluated by chaperone activity, ability to intracellular transport and in the test of activation of cytotoxic lymphocytes. Chaperone activity was 72 ± 6%. The ability to intracellular transport was estimated at 40-50% (in human U-937 human myeloid leukemia cells - at least 40%; K-562 human erythroleukemia - 49 ± 10%; mouse B16 melanoma - 44 ± 8%). Activation of cytotoxic lymphocytes in the CTL test (50 μg / ml HSP70) K-562 - 40-42%; mouse rhabdomyosarcoma RA-2 - 45-48%, rat glioblastoma C6 - 45-52%.

In the third stage, lipopolysaccharides (LPS) were removed from the purified HSP70 solution. For this purpose, the purified protein solution obtained after affinity chromatography was incubated with Polymyxin B-Agarose gel manufactured by Pierce Biotechnology in a ratio of 100 mg HSP70 - 10 ml gel. After a 30 minute incubation, the protein was separated from the sorbent by centrifugation at 500 rpm.

The concentration of bacterial LPS in the final preparation was evaluated using the test kit test E-Toxate Assay (Sigma, USA). The LPS level in the preparation was 0.4 ng / mg protein.

Claims (5)

1. The bacterial strain Escherichia coli is a producer of human heat shock protein (HSP70) obtained by transforming the strain Escherichia coli BB 1553 with the vector plasmid pMSHsp70. 2. A method of producing a recombinant human heat shock protein, comprising cultivating cells of a recombinant producer strain of Escherichia coli, isolating the recombinant protein from bacterial biomass, purifying it from bacterial protein impurities using chromatography, characterized in that the strain is cultivated as a producer strain Escherichia coli BB 1553, transformed with the vector plasmid pMSHsp70, protein isolation is carried out by repeated freezing-thawing of cells and processing the resulting suspension a solution of DNA-Zy and a solution of magnesium chloride, followed by centrifugation of a protein-containing solution and chromatographic purification of the supernatant, which is carried out sequentially on a DEAE-Sepharose ion-exchange sorbent with balanced buffer solution at pH 7.5 and affinity chromatography on ATP-agarose, after which the solution obtained after affinity chromatography, they are purified from lipopolysaccharides by incubation with Polymyxin B-Agarose gel. 3. The method according to p. 2, characterized in that the cleaning on an ion-exchange sorbent DEAE-Sepharose is carried out by pre-equilibrating the column with five volumes of buffer solution containing 20 mm Tris-HCl, 20 mm NaCl, 0.1 mm EDTA at pH 7.5 , and two volumes of the same buffer solution containing 0.1% Triton X-100 and 0.2 mM phenyl methyl sulfonyl fluoride, and elution was carried out with 1.5 volumes of 0.35 M NaCl in the buffer solution. 4. The method according to p. 2, characterized in that the purification on a column with ATP-agarose is carried out on a sorbent previously balanced with 20 volumes of buffer solution containing 20 mm Tris-HCl, 20 mm NaCl, 0.1 mm EDTA, and the elution is carried out with 3 mm adenosine triphosphate dissolved in the same buffer solution. 5. The method according to p. 2, characterized in that the purification of lipopolysaccharides is carried out by incubation of the solution of purified protein obtained with affinity chromatography with Polymyxin B-Agarose gel in a ratio of 100 mg HSP70 - 10 ml of gel.