RU2361921C1 - WAY OF RECEPTION FUNCTIONALLY ACTIVE RECOMBINANT PROTEIN OF ULCER (LF) LETHAL FACTOR, RECOMBINANT PLASMIDE DNA pETHIS-LF CODING ACTIVE PROTEIN LF; H STRAIN ESCHERICHIA COLI BL-HISLF PRODUCING ACTIVE PROTEIN OF MALIGNANT ANTHRAX LETHAL FACTOR - Google Patents

Abstract

FIELD: medicine; microbiology.

SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.

EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.

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Description

The invention relates to biotechnology, specifically to the field of genetic engineering, relates to a method for producing active recombinant anthrax lethal factor LF protein, recombinant pETHIS-LF plasmid DNA encoding an active anthrax lethal factor protein LF, and Escherichia coli bacterial strain producing an active lethal factor protein anthrax.

The lethal anthrax factor (LF), along with the protective antigen (RA), is an important component of the lethal toxin of anthrax and is the main factor determining the lethal outcome in anthrax invasion. The main areas of application of the active LF protein are biological research, biomedicine and drug development, in particular the development of diagnostic whales for early detection and pharmaceutical preparations for the treatment of anthrax infection and vaccination against anthrax. This can be important both for combating local outbreaks of the disease and for quickly neutralizing the consequences of possible bioterrorist acts using bacteria or anthrax bacteria spores.

Anthrax is an infectious disease caused by toxigenic strains of the gram-positive bacterium Bacillus anthracis. Bacterial virulence is determined by a capsule consisting of poly-D-glutamic acid and exotoxin, which includes three components - lethal factor (LF), protective antigen (RA) and swelling factor (EF). Separately, these proteins do not have a toxic effect, but combinations of LF + PA (lethal toxin) and EF + PA (puffiness toxin) lead to various pathological consequences both in mammals and humans, and in cell culture. Functionally, anthrax LF is a Zn ²⁺ -dependent metalloprotease that specifically cleaves the proteins of the kinase family of mitogenically inhibited protein kinases MAPKK (in particular, MEK1), leading to the lysis of cells exposed to the toxin under the influence of macrophages. Studies were carried out on the enzymatic activity of LF, its substrate specificity, and its three-dimensional structure.

The lethal anthrax factor is a protein with a molecular weight of 90 kDa, structurally composed of four domains with a predominantly helical structure. Domain 1 consists of 12 helical sections balancing the four-chain layer. This domain binds to a protective antigen. Domain 2 is similar to the second domain of the Bacillus cereus toxin VIP2, but does not have its catalytic radicals. Within the sequence of domain 2 is the sequence of domain 3. This domain is made up of repeats that result from duplicating the sequence originally belonging to domain 2. Domain 4 is a catalytically active protease. The beta layer and six alpha helices form a structure similar to that of another metalloprotease, thermolysin. The binding of the substrate protein of the MAPKK family occurs in the depression between domains 3 and 4. The active center of the enzyme is composed of the amino acid radical Glu ₆₈₇ behind the water molecule and the zinc atom bound to His ₆₈₆ , His ₆₉₀ , Tur ₇₂₈ and Glu ₇₃₅ radicals (Pannifer AD, Wong TY, Schwarzenbacher R, Renatus M, Petosa C, Bienkowska J, Lacy DB, Collier RJ, Park S, Leppla SH, Hanna P, Liddington RC. (2001) Nature, 414, 229-233).

A known method of isolating a recombinant lethal factor of anthrax (Gupta P, Batra S, Chopra AP, Singh Y, Bhatnagar R. (1998) Infect Immun, 66 (2), 862-865), which uses the expression construct based on the plasmid pQE30, expressed in E. coli strain SG 13009. The disadvantage of protein production using this design is that more than 40% of the protein is in a degraded state (apparently due to the cleavage of E. coli proteases), which, of course, reduces the protein yield when using this designs. A large initial degree of protein degradation leads to the fact that three stages of protein purification are required to obtain a homogeneous product. The expression construct used does not contain sequences for specific detection of the expression product.

The closest to the claimed genetic engineering method is known for producing a mutant LF protein using a pET30-based construct under the control of a phage T5 promoter in expression strains of E. coli (RF patent No. 2287581, publ. 11/20/2006). The protein thus obtained and its mutants are used to develop anthrax vaccines. However, it is known that the recombinant LF protein synthesized using the used construct has up to 40% degradation. The patented expression construct does not contain sequences for specific detection of the expression product.

The invention solves the problem of creating a producer and obtaining an active recombinant protein of the lethal factor of anthrax with high yield, low degree of degradation, easily detectable and using simplified technology using two stages of chromatographic purification instead of three.

The problem is solved by recombinant plasmid DNA pETHIS-LF, which provides the synthesis of a hybrid recombinant active protein of the lethal factor of anthrax in Escherichia coli cells, having a molecular weight of 5.15 MDa, consisting of a BamHI-NdeI DNA fragment of the plasmid pET22b (+); a synthetic sequence encoding six histidines and a c-myc peptide, as well as a BamHI / SalI DNA fragment containing a sequence of the full-length lethal factor of anthrax adapted to these sites containing the β-lactamase gene as a genetic marker that determines the resistance of cells transformed with pETHIS-LF plasmid Escherichia coli to penicillin antibiotics; unique restriction endonuclease recognition sites located to the left of the BamHI site: XhoII 657 bp, StuI 1572 bp, XhoI 1625 bp, BcoI 1625 bp.

Also due to the Escherichia coli strain BL-LFHIS producing a hybrid polypeptide containing a lethal anthrax factor fused to a sequence of six histidines and a c-myc peptide.

And also due to the method of producing a recombinant lethal factor of anthrax, which includes the transformation of Escherichia coli BL21 (DE3) cells of the expression plasmid DNA pETGST-LFmin encoding the recombinant protein of the lethal factor of anthrax, culturing the resulting strain, destroying bacterial cells by adding Triton X-100 in buffer a solution of pH 7.4 containing a protease inhibitor, followed by purification of the target product by metal-chelate chromatography and gel filtration.

The technical result of the invention is to obtain, in a purified form and with a high yield (up to 95% purity, up to 100 mg per liter of bacterial culture without fermentation), a homogeneous recombinant anthrax lethal factor protein using a simplified technology involving two stages of chromatography instead of three.

To increase the level of production and reduce the degree of degradation of the LF protein, a plasmid with the T7 polymerase promoter containing the sequence corresponding to the c-myc peptide for detecting the product and a sequence of six histidines for the efficient purification of the synthesized protein is used as the base construct. To obtain the basic vector construction of pETNHIS into plasmid pET22b (+) (Novagen) that does not contain a BglII site (the restriction site sequence is violated by processing the plasmid with BglII restriction endonuclease followed by completion of the ends with the Maple fragment of DNA polymerase I and ligation of the completed ends) at the NdeI and BamHI introduces a DNA fragment synthesized by polymerase chain reaction, encoding the sequence of six histidines and the c-myc peptide.

The full length gene of the lethal anthrax factor gene is cloned into the pETNHIS plasmid pETNHIS obtained by the described method at the BamHI and XhoI restriction endonuclease sites. sticky ends).

The resulting pETHIS-LF expression construct was transformed into E. coli strain BL21 (DE3) and expressed in 2xYT medium. The yield of recombinant protein of lethal factor of anthrax of high purity (97%) after two stages of chromatographic purification reaches 15% relative to the total protein of the cell.

The resulting product is detected with specific antibodies to the c-myc peptide.

The proposed technical solution uses a producer strain of Escherichia coli BLHIS-LF containing plasmid DNA pETHIS-LF and which is a superproducer of the recombinant protein LF.

The design of the recombinant plasmid DNA pETHIS-LF provides a high level of expression of the anthrax lethal factor gene cloned in them.

The lethal factor anthrax gene was obtained by polymerase chain reaction with the DNA of anthrax bacteria at the State Scientific Center for Applied Microbiology (FGUN SSCPMB).

The terminal sections of the gene containing the BamHI and SalI restriction endonucleases sites corresponding to the applied vector were introduced using the polymerase chain reaction with synthetic oligonucleotide primers LFBamHI, LFSalI (Fig. 1), and then the gene was cloned into the pETNHIS vector plasmid.

The proposed producer strain Escherichia coli BL-HISLF is characterized by the following features.

Morphological signs. Rod-shaped cells, non-spore, gram-negative.

Cultural signs. Bacterial cells grow well on simple nutrient media. When grown on agar, they form round, cloudy yellow-gray colonies with a slightly uneven edge, with a diameter of up to 3 mm. When growing on liquid media (LB, 2xYT) they form intense turbidity.

Physico-biological signs. Cells grow at temperatures from 4 to 40 ° C with an optimum of 37 ° C and optimal pH values from 7 to 7.5. Amino acids, carbohydrates, glycerin are used as a carbon source. Organic nitrogen (amino acids, yeast extract, tryptone, etc.) and inorganic mineral salts in ammonium form can be used as a nitrogen source.

Antibiotic resistance. Cells are resistant to penicillin antibiotics (up to 100 μg / ml).

The producer strain is obtained by transforming competent E. coli cells with the corresponding recombinant plasmid DNA.

The BL-HISLF strain is a superproducer. Upon induction with isopropylthio-β-galactoside, an effective synthesis of the target LF protein occurs, which accumulates in the cell cytoplasm in a soluble state and accounts for up to 50% of the total cell protein.

The invention is as follows. The recombinant plasmid DNA pETHIS-LF containing the sequence of the full-length anthrax lethal factor gene is constructed. For this, the full-sized gene of the lethal factor of anthrax is obtained by polymerase chain reaction with synthetic oligonucleotide primers (Fig. 1). The primers contain BamHI restriction endonuclease sites (N-terminus of the gene, LFBamHI primer) and SalI (C-terminus of the gene, LFXhoI primer), the obtained DNA is digested with the corresponding restriction endonucleases and then ligated with the BamHI and XhoI site-linked vector plasmid DNA pET. Figure 2 presents the gene sequence of the full-sized lethal factor of anthrax. Figure 3 shows the sequence of expression constructs of the plasmid pETHIS-LF.

The competent E. coli DH12S cells or other cells without DE3 lysogen and their own resistance to penicillin antibiotics are transformed with a ligase mixture and plated on 2xYT-arap containing 100 μg / ml ampicillin or another penicillin antibiotic. The resulting clones are analyzed for the presence of a plasmid containing the LF gene by polymerase chain reaction with primers LFBamHI and LFSalI. Of the clones whose PCR leads to the formation of a fragment of the desired length when analyzing reaction products by agarose gel electrophoresis, plasmid DNA is isolated by alkaline lysis. Insertion of the fragment is confirmed by plasmid DNA sequencing. The obtained recombinant plasmid DNA was transformed into competent cells of the E. coli strain BL21 (DE3), the obtained clones were analyzed for the expression level of the target protein and the producer strain was selected.

The E. coli BL-LFHIS producer strain is grown on rich medium (2 × YT, Terrific Broth, etc.) until the culture has an optical density of 0.6-1.2 OU and is induced with 1 mM isopropylthio-β-D-galactoside. Expression is carried out for 6 hours at a temperature of 25 ° C.

Cells from the expression culture are harvested by centrifugation at 5000 rpm and treated with lysozyme (0.5 mg / ml) at 4 ° C in a buffer containing 30 mM Tris-HCL pH 7.4, 150 mM NaCl supplemented with a protease inhibitor (Complete EDTA-free protease inhibitor cocktail, Roches) by adding Triton X-100 to a concentration of 0.5%. MgCl _{2 was} added to the lysate to a concentration of 5 mM and DNA was destroyed with DNase (10 μg / ml). The resulting lysate is centrifuged for 20 minutes at 18,000 g and applied to the column. The first stage of chromatographic purification is carried out on a metal-chelate sorbent. The second stage of purification is carried out on a gel filtration column in a buffer containing 30 mm Tris-Hcl pH 7.4 and 70 mm NaCl. Fractions containing purified LF are determined by denaturing polyacrylamide gel electrophoresis and frozen at -70 ° C for storage.

Product detection is carried out using an immunoblot with antibodies specific for the c-myc peptide.

The activity of the obtained recombinant lethal anthrax factor was measured using a specific substrate for the lethal anthrax factor and containing a chromotag (Lethal Factor Protease Substrate 2, Biotrend, Germany) by increasing the absorption in solution at a wavelength of 405 nm on a spectrophotometer. Kinetic measurements are carried out for 30 minutes at 37 ° C.

The invention is illustrated by graphic materials:

figure 1 - primers for amplification of the gene for lethal factor anthrax;

figure 2 - gene sequence of the full-sized lethal factor of anthrax and its fragment without domain I;

figure 3 - sequence of the expression constructs of the plasmid pETHIS-LF.

The invention is illustrated by examples.

Example 1

Construction of recombinant plasmid DNA pETNHIS-LF

The chemical synthesis of oligonucleotides is performed by the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (BIOSETS, Novosibirsk) with the oligonucleotide chain being extended from the 3 'to 5' end using protected phosphamidites (5'-dimethoxytrityl-N-acyl-2'-deoxynucleoside- 3'-O- (β-cyanethyl-diisopropylamino) phosphites) activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 μM, using porous glass (pore size 500 A) as a support, to which the first nucleoside unit (load 20-30 μM / g) is connected via a 3'-succinate bond. The synthetic cycle of the standard phosphoamidite method is used.

To prepare the DNA vector, 3 μg of the pETNHIS plasmid is treated in 20 μl of BamHI buffer (New England Biolabs) with 10 BamHI restriction endonuclease units, and then in 20 μl of buffer 2 (New England Biolabs) with 10 XhoI endonuclease units (New England Biolabs). Restriction is carried out for 1 hour at 37 ° C. A vector fragment of 6.09 kbp After electrophoresis in an agarose gel, they are placed in a dialysis bag with a maximum pore capacity of 12-14 kDa and eluted by current in a Tris-borate electrophoresis buffer, and then treated with a phenol-chloroform mixture (1: 1) and DNA is precipitated from the solution with ethanol.

To prepare a fragment of the LF gene, PCR amplification is performed using bacterial anthrax DNA 0.05 μg as a template in the sample, and synthetic oligonucleotides A and B (20 rM each) as primers. PCR is carried out in a DNA amplifier, in a buffer solution containing each of the four dNTPs at a concentration of 0.5 mM and 1 unit. Pfu polymerase activity (Promega). PCR is carried out in a mode that provides 1 minute denaturation at 95 ° C, annealing for 30 seconds at 57 ° C, elongation - 40 seconds at 72 ° C, 25 cycles of PCR. After that, the reaction mixture is deproteinized with a phenol-chloroform mixture (1: 2) and precipitated with ethanol. The precipitate was dissolved in 20 μl of water and digested with BamHI and SalI restriction endonucleases (reaction with the restriction endonuclease SalI manufactured by Fermentas is carried out in Fermentas buffer O). The target fragment is isolated by elution from an agarose gel.

0.5 μg of the obtained synthetic fragment of the recombinant lethal anthrax factor gene is added to a solution of 0.2 μg of the above vector fragment and ligated in 10 μl of buffer containing 20 mM Tris-HCl pH 7.6, 10 mM MgCl ₂ , 0.2 mM ATP, 10 mm dithiothreititis and 10 units. activity of T4 DNA ligase for 4 hours at 16 ° C.

An aliquot of the reaction mixture is used for electroporation (BTX 600 device, 129 Ohm mode, 2.5 kB) of electrocompetent E. coli DH12S cells. Transformants are plated on plates with 2xYT agar containing 50 μg / ml ampicillin. Recombinants are screened by PCR amplification under the same conditions as the preparation of a DNA fragment for cloning using a single bacterial colony containing a plasmid as a matrix. Positive clones are identified by the presence of a 2340 bp fragment when analyzing a PCR mixture by agarose gel electrophoresis. The DNA of plasmid pETNHIS-LF was isolated from the selected clones using alkaline lysis.

Example 2

Obtaining a producer strain of E. coli BL-LFHIS and determining its productivity

The E. coli BL-LFHIS producing strain is obtained by transforming electrocompetent E. coli BL21 (DE3) cells with the pETHIS-LF plasmid as described in Example 1. Single BL21 (DE3) clones carrying the pETHIS-LF plasmid are expressed in an analytical amount (according to 5 ml). For this, E. coli BL-LFHIS is grown in rich medium (2xYT, Terrific Broth, etc.) until the culture has an optical density of 0.6-1.2 OE and is induced with 1 mM isopropylthio-β-D-galactoside. Expression is carried out for 6 hours at a temperature of 25 ° C. The expression level of anthrax lethal factor protein is analyzed by denaturing polyacrylamide gel electrophoresis and immunoblot with antibodies against c-myc peptide. For electrophoresis, equal aliquots of cells of different clones are centrifuged at 5000 rpm for 5 minutes, the precipitated cells are dissolved in 100 μl of lysis buffer with bromphenol blue dye, treated for 20 seconds with ultrasound, heated for 3 minutes at 100 ° C and applied to the gel. After electrophoresis, the gel was stained with Coomassie R-250 according to a standard method and scanned using a Shimadzu CS-930 densitometer. To set up an immunoblot, the Hybond C + membrane and gel are soaked for 10 minutes in a buffer containing 38.6 mm glycine, 47.9 mm Tris base, 0.0385% sodium dodecyl sulfate, 20% methanol. The transfer is carried out at a current strength of 1 mA per cm of gel. The membrane is blocked after transfer by a 3% solution of bovine serum albumin in PBS pH 7.4 containing 0.05% Tween. The blocked membrane is incubated with c-myc antibodies (clone CRL-1725, ATCC) at room temperature for 2 hours in a PBS solution containing 0.5% BSA, and after washing the primary antibody with PBS-0.05% Tween 20, the membrane is incubated overnight with a peroxidase conjugate antibodies to murine antibodies. The washed blot is developed using a chloronaphthol peroxidase substrate. The clones giving the highest protein yield are superproducers, they are grown in rich medium (2xYT) overnight, glycerol is added up to 15% and frozen at -70 ° C for storage.

For preparative expression, E. coli BL-LFHIS superproducer strains were grown overnight at 37 ° C with 2% glucose, 10 ml of overnight culture was placed in 1000 ml of 2xYT medium containing 0.1% glucose and 50 μg / ml ampicillin for, and grown at 37 ° C to an optical density of 1 OE. The grown culture is cooled to 25 ° C. IPTG is added to 1 mM and expression is carried out for 6 hours at 25 ° C. Next, the cells are collected by centrifugation at 5000 rpm for 10 minutes and stored in the form of frozen sediments at a temperature of -70 ° C.

Example 3

Purification of the lethal factor protein obtained using the pETHIS-LF construct and determination of its activity

Cells from the expression culture are suspended in a buffer containing 30 mM Tris-HCL pH 7.4, 150 mM NaCl supplemented with a protease inhibitor (Complete EDTA-free protease inhibitor cocktail, Roches) and treated with lysozyme (0.5 mg / ml) at + 4 ° C within 30 minutes. Lyses cells by adding Triton X-100 to a concentration of 0.5%. MgCl _{2 was} added to the lysate to a concentration of 1 mM and the genomic DNA of E. coli DNAse (10 μg / ml) was destroyed. The resulting lysate is centrifuged for 20 minutes at 18,000 g and applied to a column of a metal chelate sorbent with immobilized cobalt ion (Talon, Clontech) in a buffer containing 20 mM Na ₂ HPO ₄ pH 8.3 and 70 mM NaCl. The column is washed with 10 volumes of the same buffer and the protein is eluted with a solution containing 200 mM imidazole in the same buffer. The second stage of purification is carried out on a Superdex 200 gel filtration column in a buffer containing 30 mM Tris-Hcl pH 7.4 and 70 mM NaCl. Fractions containing purified LF were determined by denaturing polyacrylamide gel electrophoresis, as described in Example 2, and frozen for storage at −70 ° C. The purity of the drug is 95% according to densitometry. The yield of the recombinant lethal factor protein is 30 mg per liter of culture after a complete purification procedure.

The proteolytic activity of the obtained preparation is evaluated by cleaving a specific substrate for a lethal anthrax factor containing a chromotag (Lethal Factor Protease Substrate 2, Biotrend, Germany), by increasing the absorption in solution at a wavelength of 405 nm on a spectrophotometer (Tesap). Kinetic measurements are carried out for 30 minutes at 37 ° C. The kinetic parameters of substrate cleavage by the lethal anthrax factor obtained are k _cat / K _M = 4.15 ± 0.7 s ^-1 μM ^-1 .

Claims (3)

1. Recombinant plasmid DNA pETHIS-LF, providing the synthesis of a hybrid recombinant active protein of the lethal factor of anthrax in Escherichia coli cells, having a molecular weight of 5.15 MDa, consisting of: BamHI-NdeI DNA fragment of the plasmid pET22b (+); a synthetic sequence encoding six histidines and a c-myc peptide, as well as a BamHI / SalI DNA fragment containing a sequence of the full-length lethal factor of anthrax adapted to these sites, containing the β-lactamase gene as a genetic marker that determines the resistance of cells transformed with pETHIS-LF plasmid Escherichia coli to penicillin antibiotics; unique recognition sites of restriction eudonucleases located to the left of the BamHI site: XhoII 657 bp, StuI 1572 bp, XhoI 1625 bp, BcoI 1625 bp. 2. The strain Escherichia coli BL-LFHIS, producing a hybrid polypeptide containing lethal anthrax factor, fused to a sequence of six histidines and peptide c-mus. 3. A method for producing a recombinant anthrax lethal factor, including transformation of Escherichia coli BL21 (DE3) cells with expression plasmid DNA pETGST-LFmin encoding a recombinant anthrax lethal factor protein, culturing the resulting strain, destroying bacterial cells by adding Triton X-100 in pH buffer 7.4, containing a protease inhibitor, followed by purification of the target product by metal-chelate chromatography and gel filtration.

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