EA035920B1 - Recombinant flagellin extraction and purification process - Google Patents

Abstract

The invention relates to molecular biology and biotechnology and may be used to produce recombinant flagellin. Provided is a method for production of active highly-purified recombinant truncated flagellin characterized by amino acid sequence SEQ ID NO: 1, the method comprises using E.coli BL21(DE3)/pET151D-TOPO_F27 producing strain subclone which contains a gene encoding such flagellin with optimized codon composition for expression in E.coli cells, ensuring target protein yield up to 45% from the total protein, cultivating such subclone cells using 0.5 mmol IPTG target gene expression inductor, cell lysing, cell pellet precipitation, protein extraction and purification from supernatant in denaturating conditions using Ni-NTA sepharose. The process allows production of 98% pure flagellin with a yield of 800 mg per liter of culture medium. The flagellin produced by the offered process exhibits high performance in test animal model when administered both before and after irradiation.

Description

The invention relates to molecular biology and biotechnology and can be used for the production of recombinant flagellin.

Humanity is susceptible to diseases caused by radiation, toxic chemicals, viruses and drug-resistant bacteria. Substances that activate endogenous pathways of the body's defense, in particular, capable of rapidly inducing the expression of cytoprotective and / or antimicrobial peptides, can be potentially effective against such diseases. The bacterial flagellum protein of bacteria, flagellin, is a powerful activator of the innate immune system. Experiments have shown that bacterial flagellin binds to TLR5 and activates NF-kB without causing systemic inflammation.

The CBLB502 polypeptide, derived from bacterial flagellin, has been shown to reduce the damage caused by irradiation in mice and primates by improving the regeneration of hematopoietic and intestinal cells. Flagellin has been shown to have a suppressive effect on intestinal cell apoptosis after exposure to radiation. The effectiveness of the use of recombinant flagellin as a radioprotector, for example, in radiation therapy of oncological diseases, and also as an effective drug in the treatment of graft versus host disease, has been shown.

In addition to radioprotective properties, flagellin, like other TLR agonists, has been shown to be effective in cancer immunotherapy experiments. Bacterial flagellin and flagellin-expressing bacteria (Salmonella) have shown an antitumor effect in models of pancreatic cancer cell metastasis to the liver and intestines. Bacterial flagellin and its derivative CBLB502 polypeptide have shown antitumor effects in in vivo models. The antitumor effect is mediated by the interaction of the flagellin molecule with the TLR5 receptor exposed on the surface of tumor cells, which leads to the synthesis of immunomodulators such as cytokines. Interestingly, agonists of the TLR5 receptor, as a rule, are less toxic than agonists of other TLR receptors (in particular, lipopolysaccharides - TRL4 agonists), which is explained by the fact that TLR5 activation does not induce the expression of cytokines such as tumor necrosis factor (TNF), interleukin 1 beta (IL1β) leading to cytokine storm and subsequent inflammation.

Flagellin, depending on the origin of a particular microorganism, differs and consists of 259-1250 amino acid residues.

Differences in the structure of flagellin are also observed among one bacterial species. Thus, most of the serovars of Salmonella enterica are biphasic, i.e. they have alternating expression of different flagellar antigens (phases). This is due to the possibility of finding in an active state only 1 of 2 flagellin genes (fliC and fljB), localized at different loci of the chromosome [Baker S., Hardy J., Sanderson K.E. et al. A novel linear plasmid mediates flagellar variation in Salmonella Typhi. PLoS Pathog, 2007, 3 (5): e59. doi: 10.1371 / journal.ppat.0030059], FliC - phase 1 flagellin (STF), fljB - phase 2 flagellin (STF2). These flagellins have the same N-terminus, homologous, differing by several amino acid residues, the C-terminus, as well as a different D3 domain.

It is known to obtain FliC using a strain based on E. coli BL21 (DE3) cells and plasmid DNA pET151D-TOPO, into which a gene encoding flagellin is introduced, which additionally encodes a polyhistidine sequence at the N-terminus for ease of purification using metal chelate chromatography, as well as a site recognition by the V5 antibody and the site of hydrolysis by proteinase TEV (RF patent No. 2524133). Recombinant flagellin makes up 37% of the total protein of the cell lysate upon induction with 0.2% lactose. Recombinant flagellin was purified from cell lysates by metal chelate chromatography followed by dialysis. The purity of the isolated flagellin was not less than 97% based on the results of polyacrylamide gel electrophoresis followed by densitometry. The study of activity was carried out on a cell line.

However, flagellin activity can also be obtained using its fragments. In the article Grebenyuk A.N. et al., 2013, described the good results of the truncated form of flagellin, represented by the FliC N- and C-terminal domains fused through the flexible bridge (Grebenyuk A.N., Aksenova N.V., Petrov A.V., Al-Shekhadat R.I., Klimov N.A., Simbirtsev A.S. Obtaining various variants of recombinant flagellin and assessing their radioprotective effectiveness. Bulletin of the Russian Military Medical Academy, - No. 3 (43), 2013, pp. 75-80). This flagellin was cloned into E. coli BL21 [DE3] Star cells (Invitrogen) using pET302 / NT-His plasmid DNA (Invitrogen). The protein yield upon induction (induced by 0.2% lactose) is not indicated. Purification was carried out using metal chelate chromatography. The study of activity was carried out on cell lines and mice.

Methods for producing flagellin, including active, in industrial quantities are not known.

The problem solved by the authors was the creation of a technology that makes it possible to obtain active recombinant flagellin in industrial quantities. The level of production for efficient industrial production according to the generally accepted parameter is not less than 20%. Thus, when optimizing the technology for producing a pharmaceutical substance, it was necessary to obtain the target protein in an amount of at least 20% of the total protein.

For this, a strain was created based on E. coli BL21 (DE3) cells and plasmid DNA pET1510TOPO, into which the gene encoding truncated flagellin was introduced, characterized sequentially

- 1,035,920 amino acids SEQ ID NO.:1, codon-optimized for expression in E. coli cells. Among the grown clones, the most productive was chosen - the one that provides the highest flagellin yield - 45%, with induction with 0.2% lactose. With further development, the protein yield was 800 mg per 1 L of the nutrient medium during cultivation in a 10 and 50 L fermenter with induction of the target protein expression with 0.5 mM IPTG. Purification using immobilized metal-affinity chromatography and Ni-NTA Sepharose sorbent under denaturing conditions resulted in a yield of 800 mg per 1 liter of nutrient medium.

The technical result from the use of the technology is to obtain an active highly purified flagellin in industrial quantities. This technical effect is achieved through the use of the most productive cell subclone of the flagellin-producing strain, including through the codon optimization of the target gene for expression in E. coli cells, the use of a suitable inductor, isolation and purification of the pharmaceutical substance from the biomass of the producer strain under denaturing conditions with using a specific sorbent.

The technical result from the use of the technology is also expressed in expanding the range of methods for producing flagellin, which is important, given the wide range of applications and the significance of this protein, and also considering that prolonged use of one protein preparation can cause a decrease in sensitivity to it in the body.

The essence of the invention

A method for producing an active highly purified recombinant flagellin characterized by the amino acid sequence SEQ ID NO.:1 is proposed, which consists in using a subclone of the E. coli BL21 (DE3) / pET151D-TOPO_F27 producing strain containing a gene encoding such a flagellin optimized by codon the composition for expression in E. coli cells, providing the target protein yield up to 45% of the total protein, cells of such a subclone are cultured using the target gene expression inductor 0.5 mM IPTG, cells are lysed, cell pellets are precipitated, protein is isolated and purified from the supernatant under denaturing conditions using Ni-NTA Sepharose.

Optimization for the codon composition for the organism of the target gene expression can be performed manually, or using specialized software, for example, at molbiol.ru, or encorbio.com/protocols/Codon.htm, based on the amino acid sequence of the protein. The sequence of the elements in the plasmid DNA is clear to the average person skilled in the art.

Used strain Escherichia coli BL21 (DE3) / pET151D-TOPO - producer of flagellin, based on Escherichia coli BL21 (DE3) cells, transformed with plasmid DNA pET151 / D-T0P0, encoding the target protein.

The technology for obtaining flagellin is described in more detail in the examples.

The authors of the present invention carried out laboratory studies confirming the possibility of implementing the described invention. The obtained research results are illustrated by the following examples.

Example 1. Creation of the most productive clone of the flagellin-producing strain 1.1. Creation of a nucleotide sequence encoding flagellin

The amino acid sequence of SEQ ID NO: 1, characterizing the truncated flagellin, was converted into a nucleotide sequence with simultaneous codon optimization for expression in E. coli cells using the program at the molbiol.ru website and adding a start and stop codon, as well as restriction sites flanking the resulting gene. The calculated nucleotide sequence was synthesized chemically using an ASM-800 DNA synthesizer (BIOSET, Russia).

1.2. Creation of plasmid DNA pET151 / D-TOPO_F27

The resulting DNA fragment, as well as the pET151 / D-TOPO vector (Invitrogen, USA), were treated with the appropriate restriction enzymes according to the instructions for these enzymes. Then the obtained fragments were purified using preparative electrophoresis and used in the ligation reaction.

A ligation reaction of the purified restriction DNA fragment into the purified restriction vector was performed. The reaction mixture contained 2 μl of purified restricted DNA fragment, 3.5 μl of 10X ligase buffer (1X buffer contains 10 mM Tris-HCl (pH 7.5 at 37 ° C), 10 mM MgCl ₂ , 50 mM NaCl, 0.1 mg / ml bovine serum albumin (BSA)) (Thermo Fisher Scientific Inc.), 3.5 μl 50% polyethylene glycol 4000, 1 μl restriction vector, 5 μl T4 ligase (Thermo Fisher Scientific Inc.). The reaction mixture was brought to 35 μL with non-nuclease water and incubated at 22 ° C for 4 h. The enzymatic reaction was stopped by incubating the reaction mixture at 65 ° C for 15 min.

The mixture was purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 μm (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.

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1.3. Creation of a strain based on Escherichia coli DH10B / R cells for amplification of the obtained plasmid DNA

Competent cells of Escherichia coli strain DH10B / R (Gibko BRL, USA) with genotype F-mcrA Δ (mrr-hsdRMS-mcrBC) <p80dlacZAM 15 AlacX74 deoR recA1 endA1 araD139 Δ (ara, leu) 7 r69 galL galU nK / and then carried out their transformation with the resulting ligase mixture.

E. coli cells were prepared for transformation with the resulting plasmid DNA - competent cells were obtained - as follows. The cells were incubated at + 37 ° C for 16 h in 5 ml of L-broth containing 1% tryptone, 1% yeast extract, and 1% sodium chloride. The culture was diluted with fresh L-broth 50-100 times and grown on a shaker at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 min. Transferred 100 ml of culture into a sterile centrifuge tube and pelleted cells at + 4 ° C at 5000g for 10 min. The supernatant was discarded, the cells were resuspended in 50 ml of 0.1 M CaCl ₂ , cooled on ice. The cells were incubated on ice for 20 min. The cells were pelleted for 10 min. at 5000 rpm, + 4 ° С. The supernatant was discarded, the cells were resuspended in 3 ml of 0.1 M CaCl ₂ , cooled on ice.

The resulting competent cells were transformed by electroporation. We used an Eporator electroporator (Eppendorf, Germany) and sterile electroporation cuvettes (Eppendorf, Germany), 100 μL, 1 mm slit.

To 12 μl of competent cells, 1 μl of a tenfold diluted ligase mixture was added, mixing and transferring to a cuvette, which was placed in an electroporator. The transformation was carried out with an electrical pulse of 1.7 kV with a duration of 5 ms. After transformation, the cells were placed in 1 ml of SOC medium (2% bactotrypton; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl ₂ , 10 mM MgSO ₄ ; 20 mM glucose) and incubated for 40 min at 37 ° С, after which it was embedded in LB-agar with antibiotic on a Petri dish and incubated for 16 h at 37 ° С.

Clones of E. coli grown on a Petri dish with ampicillin were analyzed for the presence of plasmid DNA used in the present experiment, with the simultaneous transfer of the analyzed clones of E. coli cells to separate Petri dishes with a selective medium. Plasmid DNA was isolated from such clones and analyzed by sequencing using specific primers. This made it possible to select clones-producers of the developed plasmid DNA. These strains were used to obtain plasmid DNA pET151 / D-TOPO_F27.

1.4. Obtaining plasmid DNA pET151 / D-TOPO_F27

The production and isolation of plasmid DNA was carried out as follows. A single colony of E. coli plasmid producer cells grown on LB agar in a Petri dish supplemented with ampicillin was inoculated into a standard liquid LB medium (Gibko BRL, USA), 2-10 ml, containing ampicillin at a concentration of 50 μg / ml, and carried out fermentation at 37 ° C in a thermostated rotary shaker for 16 hours at 250 rpm. The resulting culture of bacterial cells was precipitated by centrifugation at 4000g for 10 min at + 4 ° C. The supernatant was removed and plasmid DNA was isolated from the cell pellet using a kit for the isolation of plasmid DNA (Cytokin, Russia), according to the instructions for the kit. The isolated plasmid DNA was analyzed by electrophoresis in 0.8% agarose gel.

1.5. Creation of a flagellin-producing strain

The plasmid DNA pET151 / D-TOPO F27 isolated according to the above method was transformed into cells of E. coli strain BL21 (DE3) (Invitrogen, USA), with genotype F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3), containing genome / De3 lysogen and rne131 mutation. The mutated gene me (rne131) encodes a truncated form of RNase E, which reduces intracellular destruction of mRNA, leading to an increase in its enzymatic stability. Ion- and ompT-mutations in the protease genes make it possible to obtain non-proteolyzed recombinant proteins in large quantities.

Received competent cells E. coli BL21 (DE3) and transformed them with the obtained plasmid DNA as described above, instead of a tenfold diluted ligase mixture used a solution of the plasmid in water. The grown clones were checked for the presence of the target plasmid DNA using sequencing of the isolated plasmid DNA.

1.6. Identification of the most productive clone of the flagellin-producing strain

The overnight culture of the studied cells was grown in a minimal volume of LB. The next day, the overnight culture was added in a ratio of 1: 100 to 100 ml of liquid nutrient medium. The optical density was measured on a spectrophotometer at a wavelength of 600 nm. During the log phase of exponential cell growth, inoculations were made every 15 min on a solid nutrient medium with ampicillin, 100 μg / ml (selective medium) and without an antibiotic.

Constructed a curve of the dependence of the number of cells on time in semi-logarithmic coordinates. The time of doubling the number of cells (generation period) was determined from the curve. The number of cells containing the plasmid (colonies grown on the selective medium) was counted relative to the total number of cells (the number of colonies on the medium without antibiotic) before and after cell doubling. Calculated

- 3035920 the number of cells that have lost the plasmid, and compared with the total number of cells.

Clone stability was calculated based on the following mathematical model.

Let N - the number of cells containing the plasmid, n - the number of doublings (each doubling has a certain period of time). Then the number of cells at some point in time -N ⁿ

Let p be the number of cells losing the plasmid in one generation, expressed in fractions. Then the number of cells containing the plasmid is N (1-p) ⁿ .

Let's say we are interested in how many divisions half of the cells will lose the plasmid, that is, N (1-p) n = N / 2.

Hence n = log ( _1-p ) 1/2.

Thus, if the loss of the plasmid is 5% per generation, then after n = log ₀ , ₉₅ 1/2 = -13.5 divisions, half of the cells will not contain the plasmid. Minus means that there is a process of decreasing the number of plasmids in the growing mass of cells.

It was shown that the percentage of cells losing the plasmid in one generation in the logarithmic phase in different selected clones of the producer strain significantly differs. The greatest stability is recorded in clone 4.

Further, this clone was plated with a thinning streak on Petri dishes, and subclones of the E. coli BL21 (DE3) / pET151D-TOPO_F27 strain were obtained to analyze the level of expression of the target gene. In total, in the subsequent work, 4 subclones of the strain 4-4-1, 4-2, 4-3, 4-4 were used, respectively.

The expression of recombinant proteins in the selected subclones was induced as follows:

The cells were incubated at + 37 ° C on a shaker for 16 h in 10 ml of LB medium (1% tryptone, 1% yeast extract, and 1% sodium chloride). The culture was diluted with fresh LB medium 50 times and grown on a shaker at + 37 ° C until the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm. After that, the induction of expression of recombinant proteins was carried out by adding RI! 'G to the culture to a final concentration of 1 mM. It was left for 8 h to determine the level of protein expression. For this, aliquots of cells were taken 8 h after addition of IPTG. The remaining cells were concentrated by centrifugation.

Expression control was performed by disc electrophoresis of aliquots of cells after induction. Electrophoresis of cell lysates was performed in 12.5% PAGE under denaturing conditions in a heterogeneous (stepwise) buffer system (disk electrophoresis) using the isotachophoresis mechanism at the stage of sample concentration.

Cell lysates were prepared from 1 ml of cell culture in the following way: the cells were precipitated by centrifugation and resuspended in 100 μl of buffer (0.2 M Tris-HCl pH 7.5; 0.2 M NaCl; 0.01 M sodium acetate; 0.01 M β-mercaptoethanol and 5% - glycerin), then boiled for ten minutes. The resulting lysate was used to analyze the protein composition.

The obtained electropherograms were used to determine the level of expression of the target protein relative to total cellular proteins using the TotalLab computer program.

Based on the data obtained, the subclone of the producer strain 4-2 was selected for subsequent work, which provided a level of synthesis of 35% of the target protein from the total protein.

Example 2. Induction of the expression of the target gene using the obtained subclone of the flagellin producer strain

Expression induction was obtained using both IPTG and 0.2% lactose.

2.1. induction in laboratory volumes

2.1.1. upon induction of flagellin synthesis by 0.2% lactose according to the Studier method

For the cultivation of the obtained subclone of the producer strain, we used a standard agar LB medium containing ampicillin at a concentration of 50 μg / ml and glucose at a concentration of 1% to block non-specific expression.

Expression was induced when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm.

For autoinduction of expression by the Studier method, PYP-5052 medium was used, consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K ₂ HPO ₄ , 25 mM ( NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose and 0.2% lactose, 0.2% lactose was used as an inducer (Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif . 2005 May; 41 (1): 207-34).

A single colony of the producer strain (variant) was inoculated into the PYP-5052 medium containing ampicillin at a concentration of 50 μg / ml. Fermentation was carried out at + 37 ° C in a thermostated rotary shaker at 250 rpm for 14 h until there was no significant change in OD ₆₀₀ for 1 h. An aliquot was taken for analysis of the expression of the gene encoding flagellin by electrophoresis in PAGE, every hour, the end of the induction, the biomass was precipitated by centrifugation at 9000 g.

2.1.2. upon induction of flagellin synthesis by IPTG

The induction of gene expression using IPTG was carried out as follows. The cells of a single colony of the producer strain subclone were incubated at + 37 ° C in a thermostated rotary shaker at 250 rpm for 16 h in LB medium (1% tryptone, 1% yeast extract, and 1% sodium chloride), containing ampicillin at a concentration of 50 μg / ml. The culture was diluted with fresh LB medium 50 times and grown in a thermostated rotary shaker at 250 rpm + 37 ° C until the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm. Thereafter, the induction of expression of the recombinant gene was carried out by adding IPTG to the culture to a final concentration of 0.5 and 1 mM. The induction was carried out for 14 hours, a sample was taken for analysis using SDS-PAGE every hour, after the end of the induction, the biomass was precipitated by centrifugation.

2.1.3. Analysis of taken samples

The cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonyl fluoride, based on 5-7 ml of buffer per 1 g of cells. The cell suspension was sonicated 7 times for 30 s with an interval of 30 s (the ultrasound frequency is 22 kHz), a sample was taken for SDS-PAGE analysis (PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate).

The maximum expression level of the gene encoding flagellin in E. coli cells is 28 ± 0.5% upon induction with 1 mM IPTG at + 37 ° C for 3 h and 19 ± 0.7% upon induction of 0.5 mM I! IG at + 37 ° C for 3 h. Upon induction of the expression of the gene encoding flagellin by 0.2% lactose, the following result was obtained: the content of the target protein from the total cellular protein was 44.5 ± 0.5%. Thus, the most optimal mode of induction of expression of the gene encoding the target protein in cells of the E. coli subclone of strain BL21 (DE3) / pET151D-TOPO_F27, providing the maximum protein yield, was cultivation using 0.2% lactose as an inducer.

The lysate was centrifuged for 10 min at + 4 ° С, 5000 g. A sample of the supernatant fluid (supernatant) and sediment were taken for analysis of protein localization and assessment of its solubility using SDSPAGE. The analysis has demonstrated that the target protein remains soluble when cultivated using different modes and inductors, which indirectly indicates its correct (native) conformation (tertiary structure).

2.2. Scaling induction

The process of obtaining flagellin was scaled up.

2.2.1. in a volume of 10 l

For preparation, a 5-fold concentrated LB medium was used: 35 g of yeast extract, 70 g of acid hydrolyzate of casein and 35 g of sodium chloride were placed in flasks with a volume of 2 liters; distilled water was added to a volume of 1.4 l, stirred on a magnetic stirrer until the components were completely dissolved, and sterilized in an autoclave for 30 min at a pressure of 1.1 kgf / cm ² .

A laboratory fermenter of Sayany (Biosset) with a working volume of 10 liters was prepared according to the instructions for use. In the fermenter vessel, in compliance with the rules of asepsis, pH and pO ₂ sensors were installed and sealed. A compressed air supply line from a compressor with a sterilizing filter was connected to the barbater. The tap on the highway was closed. The control program was activated on the computer.

Observing the rules of asepsis, 1,400 l of concentrated 5-fold LB broth, 35 ml of ampicillin solution, sterile distilled water up to 8 l were poured into the fermenter vessel, the lid was closed, the motor of the magnetic stirrer was installed. Using the control program, the turbine rotation speed was brought to (1200-1500) rpm and the medium was stirred for 3-5 minutes, after which the engine was turned off, removed, the lid was slightly opened, and 315 ml of seed was poured into the fermenter container.

The lid of the fermenter was sealed, the hose of the peristaltic pump was connected to the technological hole in the lid, the hose was connected to a pressure gauge to measure the pressure in the fermenter, and a hose was connected to the spray catcher to remove the blown air. Using the control program, the heating of the fermenter was switched on, the temperature was automatically controlled within the range of (37.0 ± 0.5) ° C, and the turbine rotation speed was brought to 1200-1500 rpm. Hoses from the cryostat of the flow cooling system were connected to the trap on the fermenter lid, and the cryostat was turned on. The pH of the medium in the fermenter was controlled: the pH value should have been in the range of 6.8-7.2. If necessary, a solution of HCl or NaOH was fed through the technological opening in the lid using a peristaltic pump until the specified pH value was reached.

The compressor was turned on, the air pressure in the system was set to 0.20 atm. The pO2 value was monitored. The pO2 value was maintained by gradually increasing the compressed air pressure. The air flow rate was controlled by a rotameter, adjusting it with a valve at the trap outlet. The air flow had to remain within (10-15) l / min. If it was impossible to maintain the required pO2 value, oxygen was supplied to the compressed air supply system through a tee from a cylinder with a reducer.

Cultivation was continued while monitoring the pH value and optical density of the culture. For

- 5 035920 measurements of the optical density of the culture 2 h after inoculation and then every 30 min open the sampler valve on the fermenter and take samples of the culture. Measurement of optical density at a wavelength of 600 nm (A6 ₀₀ ) was carried out using an Ultraspec-10 densitometer. Aliquots of the culture were saved for subsequent control of protein content using the method of disk electrophoresis. Fermentation was carried out for 3-9 hours (with induction of IPTG), 20 hours (with induction with 0.2% lactose) after reaching OD ₆₀₀ 6 optical units.

Expression was induced when the cell culture reached an optical density of 0.650.82 optical units at a wavelength of 600 nm. The expression of the gene encoding the target protein was induced by using two modes: the induction of expression with IPTG at various concentrations and 0.2% lactose was performed.

2.2.1.1. IPTG induction

IPTG at concentrations of 1, 0.5, and 0.1 mM was used as an expression inducer. The level of expression of the gene encoding the target protein was optimized, while the induction time was varied.

In the first case, IPTG (Sigma, USA) was not added to the fermenter (the description of fermentation is above, this is a control), in the second case, IPTG was added to a final concentration of 1 mM, in the third - up to 0.5 mM, in the fourth - up to 0.1 mM, fermentation was carried out within 9 hours, every hour, aliquots of cells (1 ml) were taken to measure OD ₆₀₀ (200 μl from 1 ml), analysis of proteins using the method of disk electrophoresis (800 μl from 1 ml).

Electrophoresis of cell lysates was performed in 12.5% PAGE under denaturing conditions in a heterogeneous (stepwise) buffer system (disk electrophoresis) using the isotachophoresis mechanism at the stage of sample concentration (Laemmli, 1970).

Cell lysates were prepared from 800 μl of cell culture in the following way: cells were pelleted by centrifugation, resuspended in 200 μl of buffer (0.2 M Tris-HCl, pH 7.5, 0.2 M NaCl, 0.01 M sodium acetate, 0.01 M β-mercaptoethanol, and 5% glycerol ), boiled for 2 min. The resulting lysate was used to analyze the protein composition.

It was shown that the maximum expression level of the gene encoding flagellin upon induction of IPTG at a concentration of 1 mM is observed after 4-hour fermentation, 0.5 and 0.1 mM - 6. In this case, the protein yield is 28 ± 0.5%, 30 ± 0.8% and 19 ± 0.7%, respectively, of the total cellular protein according to densitometric analysis (Total Lab software). Without induction, none of the strains showed unintended synthesis of the target protein, leakage of the T7 promoter, which is often observed in this expression system (Grossman, 1998).

2.2.1.2. Induction with 0.2% lactose

Induction of expression was carried out by the method of Studier (Studier, 2005). The medium used was PYP5052, consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K2HPO4, 25 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose and 0.2% lactose.

A single colony of the producer strain was inoculated into PYP-5052 medium containing ampicillin at a concentration of 100 μg / ml. Cultivation and induction of expression were carried out in a 10 L fermenter until there was no significant change in OD600 for 1 h. An aliquot of cells was taken for analysis by electrophoresis, and the remaining biomass was subjected to centrifugation at 9000g.

Electrophoresis of cell lysates prepared according to the method described above was carried out according to the method described above.

The protein yield was 35 ± 0.5% of the total protein, which exceeded the values obtained upon induction of the expression of the target gene by IPTG.

Despite the fact that the highest protein yield was observed upon induction of its synthesis by lactose, when using batch cultivation, it is more advantageous to use an inductor that requires less time for growing. It is also important to consider the consumption of reagents. In this regard, during the cultivation of the Escherichia coli strain to obtain recombinant flagellin, the method of fed-batch cultures and 0.5 mM IPTG as an inducer was used. Fed culture has proven to be the most effective way to achieve high cell density and high productivity.

The cultivation was stopped, the drain valve on the fermenter was opened, the culture was poured into a container with a capacity of 10 L, the lid was closed, and the container with the culture was left in the refrigerator at a temperature of (4-7) ° C.

They turned off and removed the turbine engine, turned off the compressor, turned off the trap cooling cryostat, disconnected all hoses, and removed the fermenter lid. The fermenter vessel was sequentially washed with two 8 L portions of distilled water, the wash water was poured into two 10 L polypropylene canisters, a hydrogen peroxide solution (30%) was added to each canister, the canister lids were closed, and their contents were thoroughly mixed. The canisters were left at room temperature for 12-16 hours. 9 L of distilled water, 1 L of 30% hydrogen peroxide solution were poured into the fermenter, the lid was closed, and the turbine of the fermenter was turned on (1000 rpm, 5 min). Then the turbine of the fermenter was stopped and the fermenter was left to sterilize for 24 hours.

2.2.2. in a volume of 50 l

The E. coli strain BL21 (DE3) / pET151D-TOPO_F27 was sterilely subcultured onto slants of EDTA-containing agar and kept in a thermostat for 1 day. To obtain an inoculum, the culture was washed off the stocks, inoculated into a liquid medium with EDTA, and cultured for 2 days. After that, the inoculum in an amount of 10 ml was transferred into sterile 750-ml flasks with 200 ml of sterile liquid medium and cultured for 10 days on a shaker at 150-200 rpm at a temperature of 28-30 ° C.

Liquid nutrient medium (MgSO ₄ -7 H2O 10 ml / l, CaCl ₂ -2 H ₂ O 20 ml / l, KH ₂ PO ₄ + NaH ₂ PO ₄ -12 H ₂ O 10 ml / l, EDTA 10 ml / l , microelements 1 ml / l; adjusted to pH = 4.2; + 5.6 g / l EDTA, FeCl ₂ -4 H ₂ O 1.5 g / l, H3BO3 0.06 g / l, MnCl2-6 H2O 0.1 g / l, CaCl2-6 H2O 0.12 g / l, ZnCl ₂ 0.07 g / l, NiCl ₂ -6 H2O 0.025 g / l, CuCl ₂ -2 H ₂ O 0.015 g / l, Na ₂ MoCl ₄ 0.025 g / l; Vitamins: Pyridoxine 20 mg, Thiamine 10 mg, Riboflavin 10 mg, Nicotinic acid 10 mg, P - aminobenzoic acid 10 mg, Lipoic acid 10 mg, Nicotinamide 10 mg, Vitamin B12 10 mg, Biotin 4 mg, folic acid 4 mg Dissolved all components in 200 ml of water, sterilized at 0.5 ATM for 30 min and added to a liquid nutrient medium in an amount of 1 ml / l), used to obtain inoculum and for batch cultivation.

Solid nutrient medium was used to obtain a fresh culture of the strain. It was prepared as a liquid with the addition of 3% agar.

Since the highest protein yield was observed in batch cultivation with induction of expression of 0.5 mM IPTG, this inducer and the described fermentation mode were used to produce the target protein.

When the optical density (OD ₆₀₀ ) of the E. coli cell culture reached 6.0 OE, an expression inducer - 0.5 mM IPTG was added. Induction of expression was carried out for 12 h, with periodic supply of the necessary elements, after which the cells were precipitated by centrifugation at 13000 g for 6 min at a temperature of 10 ° C. The cell sediment was weighed.

The cells were resuspended in 30 ml of buffer (50 mM Tris HCl, pH, 50 mM EDTA, 20 mM Lcysteine, pH8.6) and disrupted using ultrasound.

After cultivation of the subclone of the target protein producer strain and induction of expression of the gene encoding this protein, 0.5 mM IPTG in a 50 L fermenter, the E. coli BL21 (DE3) / pET151D-TOPO_F27 cell culture was cooled, transferred to centrifuge beakers and precipitated by centrifugation at 2000g. The level of expression of the target gene was checked using electrophoresis followed by densitometric scanning of the electrophoretogram. The resulting biomass was resuspended in a buffer of the following composition: 50 mM Tris-HCl pH 8.6, 50 mM EDTA, 20 mM L-cysteine in a volume of 2 liters.

Lysis of bacterial masses was carried out: cells were destroyed using ultrasound (sonication time - 10 min, pulse time - 30 s, pause time between pulses - 30 s, amplitude - 70%). After the destruction of the cells, the suspension was precipitated using centrifugation at 30,000 g for 20 min at a temperature of 10 ° C.

Checking the completeness of cell lysis was carried out in the following way. An aliquot of the lysate (2 ml) was taken, added to the LB culture medium containing 1% glucose, in a volume of 100 ml, and incubated at 37 ° C in a thermostatically controlled shaker for 6 h. The culture medium remained transparent and had an optical density of less than 0.025 at a wavelength of 600 nm, which confirms the completeness of the lysis.

Thus, the protein yield was 800 mg per 1 L of nutrient medium during cultivation in a 50 L fermenter with induction of the target gene expression with 0.5 mM IPTG.

Example 3. Isolation and purification of flagellin

The sorbent capacity affects the final protein yield. As sorbents, NiNTA Agarose (Ni-NTA Sepharose) from QIAGEN, catalog number 30230, ProBond ™ Nickel-Chelating Resin from Invitrogen, catalog number R801-15, Ni Sepharose 6 Fast Flow from Amersham Bioscience were tested.

The rationale for the choice of these particular sorbents lies in the fact that in the purification technology, the compositions of the reagents used for washing the proteins are suitable for the charges of the particles of these sorbents, and only the sorbents can be varied, keeping the overall technology identical.

Was carried out purification of flagellin from lysates of E. coli cells strain BL21 (DE3) / pET151DTOPO_F27 by immobilized metal affinity chromatography (IMAC) using a sorbent Ni-NTA Sepharose. Binding to this sorbent occurs due to 6 histidine residues present at the N-terminus of the resulting recombinant protein. The protein purification procedure was carried out under native and denaturing conditions.

To purify the synthesized protein under native conditions, the biomass of E. coli cells of strain BL21 (DE3) / pET151D-TOPO_F27 was destroyed using a buffer containing imidazole, Triton X-100, Tween 20, lysozyme and DNase 1. After destruction, the remains of bacterial cells were precipitated by centrifugation, the supernatant was filtered through a 0.22 µm PVDF filter. Next, the Ni-NTA Sepharose sorbent equilibrated with a buffer for destruction was added to the resulting filtrate

- 7,035,920 cells without enzymes, assuming that 1 ml of the sorbent binds at least 40 mg of protein, and the binding was carried out with constant stirring for 2 hours. After that, the sorbent was precipitated by centrifugation and in a minimum volume was applied to a gravity chromatographic column. Elution was carried out with a stepwise gradient of imidazole (40, 100, 150, 200, and 300 mM), with a volume of elution solution equal to 10 column volumes per each step. The protein yield and purity were monitored by disk electrophoresis and by measuring the protein concentration by the Bradford method.

The flagellin protein is eluted from the column with a blurred peak: it begins to disappear at an imidazole concentration of 40 mM, reaches a maximum at an imidazole concentration of 100 mM, and ends elution at an imidazole concentration of 150 mM. In this case, the purity of the protein in the major fractions eluted with 100 mM imidazole was 80% of the impurity proteins of bacterial cells.

To purify the target protein under denaturing conditions, the biomass of E. coli cells of the BL21 (DE3) / pET151D-TOPO_F27 strain was destroyed using a buffer containing 6M guanidine hydrochloride. After destruction, the remains of bacterial cells were precipitated by centrifugation, the supernatant was filtered through a PVDF filter with a pore diameter of 0.22 μm. To the obtained protein solution, 600 ml of Ni-NTA-FF sorbent was added (the particle diameter of the sorbent is 165 μm, the binding capacity is 1 ml of the sorbent binds 40 mg of the recombinant protein, 1 ml of the sorbent has 6x10 ¹⁴ binding sites), equilibrated with a buffer for cell destruction, from calculation that 1 ml of sorbent binds at least 40 mg of protein. Sorption was carried out with constant stirring for 2 h. After that, the sorbent was precipitated by centrifugation and applied in a minimal volume to a gravity chromatographic column or washed using a stepwise pH gradient with a buffer containing 8M urea without being applied to a gravity column. In the first case, the sorbent was washed with a tenfold volume of a buffer of the following composition: 10 mM Tris-HCL pH 7.8, then with a tenfold volume of a buffer of the following composition: 10 mM Tris-HCL pH 6.0. In the second case, elution was carried out with a buffer containing 10 mM Tris-HCl pH 8.6 100 mM imidazole, each step consisted of at least two washes, 10 column volumes each. Elution was performed with a buffer with pH 3.8-4.

The protein yield and purity were monitored by disk electrophoresis and by measuring the protein concentration by the Bradford method.

When cleaning in native conditions, i.e. without destroying the tertiary structure of the protein, with a good yield in one chromatographic cycle, the target protein with a purity of 80% was obtained, the protein yield was 5 mg per liter of cell culture.

As a result of the purification of the synthesized protein under denaturing conditions, i.e. with the destruction of the tertiary structure of the protein, with a good yield in one chromatographic cycle, it was possible to get rid of all impurity proteins of the producer strain in one chromatographic cycle using only one type of sorbent - Ni-NTA sepharose. According to the densitometric analysis of the obtained electropherogram, the purity of the isolated and purified protein was 98%. The amount of protein isolated and purified from 50 ml of bacteria culture was determined by the Bradford method and amounted to 40 mg of protein, which corresponds to a yield of 600 mg per 1 liter of culture.

The highest protein yield was observed when using the Ni-NTA Agarose sorbent from QIAGEN (600 ± 25 mg per 1 L of culture). When using ProBond ™ Nickel-Chelating Resin, the protein yield was 300 ± 42 mg per liter of culture, when using Ni Sepharose 6 Fast Flow sorbent, the protein yield was 420 ± 12 mg.

Example 4. Obtaining flagellin in industrial quantities

The cultivation was carried out in a 50 L fermenter. Three cycles (series) were carried out to develop samples of a pharmaceutical substance. The operating time was carried out according to the following scheme.

Sowing of the subclone of the producer strain from the working museum was carried out. Cells of the Escherichia coli strain, a producer of flagellin, were plated with a microbiological loop from cryovials onto the surface of a Petri dish containing LB agar supplemented with 100 μg / ml ampicillin. After incubation in a thermostat for 18 h, the grown colonies were used as inoculum.

The material of 5 colonies grown on a Petri dish was transferred using a microbiological loop from the agar surface into a sterile conical flask with a capacity of 1000 cm ³ , into which 150.0 cm ^{3 of} sterile LB medium containing 100 μg / cm ³ ampicillin was placed. The flask was placed in an orbital shaker and incubated in it at a temperature of + 37 ° C and a platform rotation speed of 250 rpm. The resulting culture was inoculated in a 50 L fermenter. The synthesis of the target protein was induced using 0.5 mM IPTG. Fermentation lasted 12 hours with batch cultivation. The optical density of the biomass after cultivation was determined in a cuvette using a spectrophotometer, and it was 16 optical units at a wavelength of 600 nm.

Concentration of the biomass was carried out. Centrifugation at 2000 g yielded 1.5 L of concentrated biomass, which contained 90 g of cells per wet weight. Using electrophoresis in PAGE (12.5%), the level of expression of recombinant proteins in the producer strains was checked. It made up 35% of the total bacterial proteins. This proportion was determined using denzitometric scanning electrophoretograms. The resulting biomass was resuspended in the buffer of the following composition: 50 mM Tris-HCl pH 8.6, 50 mM EDTA, 20 mM L-cysteine in a volume of 1 liter.

The lysis of the bacterial mass was performed by sonicizing for 30 s pulse, 30 s pause, amplitude 70% (from 750 W), time 10 min. A Sonics Vibra Cell 750 instrument was used.

The completeness of cell lysis was tested in two independent ways. An aliquot of 2 ml of lysate was taken, added to the LB culture medium containing 1% glucose, 100 ml in volume, and incubated at 37 ° C in a thermostated shaker for 6 h. The culture medium remained transparent and had an optical density of less than 0.025 at a wavelength of 600 nm.

The resulting lysate was centrifuged at 20,000 g for an hour. The supernatant was collected and electrophoresis according to Lemmli was carried out with a 20 μl sample. Only E. coli proteins were visible on electrophoresis, and the fraction corresponding to the expression of each recombinant protein was missing.

For purification of recombinant flagellin, IMAC was used under denaturing conditions. The Ni-NTA Sepharose sorbent (GE Helthcare, USA), washed several times in a buffer for cell destruction, was added to the supernatant obtained after the precipitation of cell debris destroyed under denaturing conditions, on the basis that 1 ml of the sorbent binds 40 mg of protein. The binding was carried out at room temperature in a thermostated rotary shaker at 200 rpm. After that, the sorbent was precipitated by centrifugation at 600 g, resuspended in a small volume of buffer to destroy cells without using enzymes, and applied to a gravity column. The sorbent was washed with a stepwise pH gradient (7.8, 6.0, and 5.3) in a buffer containing 20 mM K-Na phosphate buffer, 500 mM NaCl, and 8M urea. Elution of protein from the sorbent was performed using a buffer containing 20 mM K-Na phosphate buffer, 500 mM NaCl, and 8M urea, pH 3.8-4.0. The obtained fractions were analyzed by disk electrophoresis in SDS-PAGE, the protein concentration in them was determined by the Bradford method.

As a result of the purification of the target recombinant protein under denaturing conditions, it was possible to get rid of all the impurity proteins of the producer strain in one chromatographic cycle using only one type of sorbent - Ni-NTA sepharose. According to the densitometric analysis of the obtained electropherogram, the purity of the isolated and purified protein was 98%. The amount of protein isolated and purified from the culture of bacteria was determined by the Bradford method, the yield was 800 mg per 1 liter of culture.

The homogeneity of the obtained pharmaceutical substance of the target protein was checked by HPLC (High Performance Liquid Chromatography).

The chromatographic homogeneity of the flagellin preparation was checked by reversed phase chromatography (RPC) on a P20 RPC HR 16/10 column (Pharmacia, Sweden) in HPLC mode. The column load was 1.0 mg. Elution was carried out with a concentration gradient of acetonitrile in trifluoroacetic acid (TFA) at room temperature (solution A = 0.1% TFA, solution B-acetonitrile in 0.1% TFA). The registration of the protein in the eluate was carried out by the absorption at 280 nm.

Chromatography showed that the obtained pharmaceutical substance of flagellin is homogeneous, since the protein is released during RPC in one peak, the protein purity reaches 99.88% for sample number 1, 100% for sample number 2 and 100% for sample number 3.

Example 5. Analysis of the activity of the resulting flagellin

The analysis of the activity of the obtained flagellin was carried out on white outbred rats weighing 200 g and aged 5 months, which corresponds to in-depth tests (Karkishchenko; 2007).

The model based on rats is preferable, since most of the literature data are available for this model - based on rats, and therefore the obtained data are easier to compare with the data available in the literature (per 1000 publications on experiments with rats - 240 publications with mice and 12 publications with hamsters). The second substantiation of the rat model is the ability of the organism of this animal to withstand lower doses of radiation than in a mouse or hamster (in a rat the half-lethal dose is 7.5 gray, in a mouse and a hamster - 9), therefore the effect of the drug has more reliable values if it is discovered. In addition, many of the available radiation installations give a dose of no more than 8 gray. Thus, the use of rats is more preferable for this third, technical reason.

For the experiment, we used adult male rats of the Wistar line, 180-190 g. The experimental animals came from the Research and Production Enterprise Nursery of laboratory animals of the FIBC RAS. The animals were kept in a vivarium in compliance with the regimen of food and water intake. The duration of quarantine (acclimatization period) for all animals was 14 days. During the quarantine, each animal was examined daily (behavior and general condition), twice a day, animals were observed in cages (morbidity and mortality). Before the start of the study, animals meeting the criteria for inclusion in the experiment were assigned to groups (experiment and control) using the method of randomization. Animals that did not meet the criteria were excluded from the study during quarantine. The animal cages were placed in separate rooms. Light mode: 12 h - light, 12 h - darkness. The air temperature was maintained within the range of 19-25 ° С, the relative humidity - 50-70%. Air temperature and humidity were recorded daily. When the weather conditions changed, the air exchange in the room was controlled using

- 9 035920 anemometer and by measuring the content of carbon dioxide and ammonia in the air. The ventilation mode was set, providing about 15 volumes of the room per hour, CO2 concentration not exceeding

0.15 vol.%, Ammonia - no more than 0.001 mg / l. The animals received water ad libitum.

As a source of radiation exposure, a Gamma-panorama device with modifications was used, mounted in special protective conditions. The radiation source is 137 _z Cs, sealed in a double ampoule made of stainless steel and located behind the protective screen in the inoperative position of the installation.

A dosage of 8.0 Gy was worked out as an irradiation mode, and tests were carried out on test groups of animals in order to test the selected model. The tests were preliminary in nature, and the goal was to find out the dose of the flagellin (PL) drug and the working ranges of parameters in the experiment. The results are presented in the table.

As can be seen from the table, the resulting flagellin preparation is active - effective both when applied before and after irradiation.

Survival of animals after irradiation (8.0 Gy, LD _80-90 ).

Animal groups N 30 days after irradiation PL 15-30 'before irradiation 1 PL at a dose of 2.0 mg / kg 15 13 2 PL at a dose of 1.0 mg / kg 15 8 3 PL at a dose of 0.5 mg / kg 15 nine 4 PL at a dose of 0.2 mg / kg 15 ten five Control 15 3 PL 15-30 'after irradiation 6 PL at a dose of 2.0 mg / kg 12 8 7 PL at a dose of 1.0 mg / kg 12 8 8 PL at a dose of 0.5 mg / kg 13 7 nine PL at a dose of 0.2 mg / kg fourteen 3 ten Control 8 1

CLAIM

Claims (1)

CLAIM A method of obtaining an active highly purified recombinant flagellin characterized by the amino acid sequence SEQ ID NO: 1, comprising using a subclone of the producer strain E. coH BL21 (DE3) / pET151D-TOPO_F27, containing a gene encoding such a flagellin, optimized in codon composition for expression in E. coli cells, providing the target protein yield up to 45% of the total protein, cells of such a subclone are cultured using the target gene expression inductor 0.5 mM IPTG, cells are lysed, cell sediments are precipitated, protein is isolated and purified from the supernatant in denaturing conditions using Ni-NTA Sepharose.