RU2580031C2 - PLASMID VECTOR pET-His8-TrxL-Acip1 STRAIN OF BACTERIA Escherichia coli BL21(DE3)/pET-His8-TrxL-Acip1 FOR EXPRESSION OF ANTIMICROBIAL PEPTIDE ACIPENSINA-1 AND METHOD OF PRODUCING SAID PEPTIDE - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of proteins, and can be used for production of Antimicrobial Peptide acipensine-1 of Russian sturgeon Acipenser gueldenstaedtii. Constructed plasmid vector pET-His8-TrxL-Acip1 acipensine-1 expression in Escherichia coli cells as a part of hybrid protein His8-TrxL-Acip1. Transformed data vector of parental Escherichia coli BL21 (DE3) and strain-producer of hybrid protein His8-TrxL-Acip1. That is followed by culturing the strain producer cell lysis is performed, affine purification of hybrid protein His8-TrxL-Acip1 on metalchelate carrier, hybrid protein splitting His8-TrxL-Acip1 cyan at residue of methionine, inserted between the sequences acipensine-1 and thioredoxin, repeated performing cleaning and final purification of the target peptide by reverse-phase HPLC.

EFFECT: invention allows to produce a biologically active acipensine-1 using a simple technique.

3 cl, 4 dwg, 4 ex

Description

The invention relates to the field of biotechnology, namely to the production of acipensin-1, an antimicrobial peptide of the Russian sturgeon Acipenser gueldenstaedtii, which can be used in medical and veterinary practice as a broad-spectrum antibiotic.

Antimicrobial peptides (AMPs) of the innate immunity system play an important role in the anti-infection protection of humans and animals [Kokryakov V.N. Essays on innate immunity. St. Petersburg: Nauka, 2006.261 s.]. These molecular factors of innate immunity occupy a special place in providing protective functions in lower vertebrates (fish, amphibians), since the adaptive immunity system in poikilothermic animals cannot provide a sufficiently fast and effective response (antibody formation) to infection at low ambient temperatures. Acipensin-1 (SEQ ID No: 1) is one of the fragments of the N-terminal part of histone H2A isolated from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii [Shamova OV, Orlov DS, Balandin SV, Shramova EI, Tsvetkova EV, Panteleev PV, Leonova Yu. F., Tagaev AA, Kokryakov VN, Ovchinnikova TV Acipensins - Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii // Acta Naturae. - 2014 .-- Vol. 6 .-- 4 (23). - R. 99-109]. In its primary structure, it is close to the antimicrobial peptides hipposin and buforin I. Acipensin-1 has a wide spectrum of bactericidal and fungicidal action, which is partially preserved in solutions with a high concentration of salts. The main target of the action of the peptide in concentrations close to the MIC is not bacterial membranes, but intracellular components. Activity against methicillin-resistant strain of Staphylococcus aureus ATCC 33591 creates the prerequisites for the use of acipensin-1-based drugs in surgery and transplantology. Like other natural AMP - derivatives of histone H2A, acipensin-1 does not show significant cytotoxic activity in relation to cultured human cells. The presence of such properties opens up prospects for the practical use of the peptide in antitumor therapy, as vectors for the delivery of drugs to malignant cells.

The only known method for producing acipensin-1 includes the steps of obtaining leukocyte mass from the blood of the Russian sturgeon Acipenser gueldenstaedtii, acid extraction of proteins and peptides, ultrafiltration, preparative polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography [Shamova OV, Orlov D.S. ., Ovchinnikova T.V., Sal H.G., Tveryanovich I.A., Popova V.A., Dyubin V.A., Kokryakov V.N. // Basic research. 2006. No1. S. 10-13]. The disadvantage of this method is the need to process large volumes of sturgeon blood to obtain the peptide in quantities necessary for its use in medical and veterinary practice.

The invention solves the problem of expanding the range of biologically active peptides and obtaining the antimicrobial peptide acipensin-1.

The problem is solved by constructing an expressing plasmid vector pET-His8-TrxL-Acip1, consisting of two DNA fragments:

- BglII / XhoI fragment with the nucleotide sequence of SEQ ID No. 2, which contains a T7 RNA polymerase transcription promoter, a lac operator, a ribosome binding site and an affinity tag coding region, a modified carrier protein thioredoxin A Escherichia coli (TrxL) and the antimicrobial peptide acipensin-1;

- BglII / XhoI fragment of plasmid pET31b (+), which contains the T7 RNA polymerase transcription terminator, replication initiation site, β-lactamase gene and lac-penpeccopa gene (lacI);

due to the producer strain of the His8-TrxL-Acip1 fusion protein by transforming the Escherichia coli strain BL21 (DE3) with the indicated vector pET-His8-TrxL-Acip1;

as well as due to the method of producing the acipensin-1 antimicrobial peptide, including culturing the producer strain Escherichia coli BL21 (DE3) / pET-His8-TrxL-Acip1, cell disruption, affinity purification of His8-TrxL-Acip1 fusion protein on a metal chelate carrier, cleavage of the hybrid protein His8-TrxL-Acip1 with cyanogen bromide according to the methionine residue introduced between the sequences of acipensin-1 and thioredoxin, repeated purification on a metal chelate carrier and purification of the target peptide by reverse phase HPLC.

In order to prevent premature death of the producer microorganism due to the expression of antimicrobial peptides in its cells and the associated loss of productivity, it is necessary to temporarily neutralize the toxicity of the synthesized product. The claimed plasmid vector pET-His8-TrxL-Acip1 provides a high level of acipensin-1 expression in Escherichia coli cells by including its amino acid sequence in the His8-TrxL-Acip1 fusion protein (SEQ ID No. 3) carrying the sequence of thioredoxin A. Thioredoxin able to accumulate in a high concentration (up to 40%) in the cytoplasm of E. coli in soluble form and is used for overexpression of biologically active polypeptides [LaVallie ER et al. (1993) Nature BioTechnology. 11: 187-193]. The need to use a carrier protein is due not only to the toxicity of mature acipensin-1 for a bacterial cell, but also to the possibility of its degradation in a heterologous system. To release the target peptide from the His8-TrxL-Acip1 fusion protein molecule, a methionine residue is introduced between the acipensin-1 and thioredoxin sequences, allowing selective cleavage of the fusion polypeptide with bromine cyan.

When splitting the TrxL-Acip1 fusion protein with cyanogen bromide, it is advisable to use Escherichia coli modified thioredoxin A as a partner for heterologous expression, which contains replacing the internal methionine residue with a residue of another amino acid, for example, leucine (M37L). It is known that such a slight change in the primary structure does not affect the spatial structure and properties of thioredoxin [Rudresh et al. (2002) Protein Eng. 15 (8): 627-33]. The absence of internal methionine residues in the carrier protein sequence reduces the number of individual polypeptide fragments resulting from a reaction with cyanogen bromide. In a preferred case, the cleavage of the hybrid protein gives only one by-product, significantly different in physicochemical properties from the target peptide, which facilitates the process of purification of the latter.

Purification of the target peptide is simplified by the inclusion of an affinity tag, an amino acid sequence that allows purification of the hybrid polypeptide by affinity chromatography on sorbents with immobilized (chelated) metal ions, as part of the His8-TrxL-Acip1 fusion protein. metal chelate cleaning. As such a sequence, a sequence of 4-10 histidine residues is usually used, most often a sequence of six histidine residues, which is introduced into the N-terminal or C-terminal region of the fusion protein or in the region separating the carrier protein and the target protein [Terpe K . (2003) Appl Microbiol Biotechnol. 60 (5): 523-33]. HAT and 6xHN fragments are also used as affinity tags for metal chelate purification [U.S. Pat. No. 7,176,298].

The composition of the claimed vector pET-His8-TrxL-Acip1 includes regulatory elements that control the expression of the hybrid protein: promoter and terminator for RNA polymerase of the bacteriophage T7, lac-operator, consensus bacterial ribosome binding site, start and stop codons. To suppress the basal expression of the fusion protein gene, the pET-His8-TrxL-Acip1 vector includes the lac-penpeccopa (lacI) gene. The claimed vector includes the replication initiation site (Ori) of the pBR322 plasmid and the β-lactamase marker gene, which determines the resistance of Escherichia coli cells transformed with the pET-His8-TrxL-Acip1 plasmid to ampicillin and allows the selection of cells containing vector DNA by growing on the corresponding selective nutrient medium.

The construction of the plasmid vector pET-His8-TrxL-Acip1 expressing the His8-TrxL-Acip1 fusion protein containing the acipensin-1 sequence can be carried out by ligation of the BglII / XhoI fragment of plasmid pET-31b (+) (Novagen) containing the initiation region replication, T7 terminator, β-lactamase and lacI genes, with an insert encoding a fusion protein gene. An artificial gene that encodes a fusion protein can be obtained by chemical synthesis of a set of oligonucleotide fragments, followed by assembly and amplification of the intermediate and final products using polymerase chain reaction (PCR). The choice of the structure of oligonucleotide primers for the synthesis of each of the structural elements (promoter, operator, ribosome binding site, thioredoxin, affinity sequence, acipensin-1) is based on data on their nucleotide sequences available from open sources (GenBank, EMBL-Bank, DDBJ data banks ) The matrix for amplification of the thioredoxin sequence is the bacterial genome or plasmid pET-32a (+) (Novagen). The replacement of the methionine codon in the composition of thioredoxin (M37L), if necessary, is carried out at the stage of assembly by the method of directed mutagenesis using PCR. Before ligation, the purified amplicon and plasmid vector are treated with restrictase enzymes to obtain sticky ends. The ligase reaction products transform competent cells of the Escherichia coli strain with the DNA recombination and restriction system turned off, for example, DH5α, DH10B or XL1-Blue. The selection of clones containing the plasmid vector with the insert is carried out using PCR and restriction analysis of the isolated vector DNA. The correct assembly of the construct is determined by vector DNA sequencing.

The producer strain of the His8-TrxL-Acip1 fusion protein containing the sequence of the acipensin-1 target peptide is obtained by transforming pET-His8-TrxL-Acip1 vector DNA preparation of competent Escherichia coli cells and selecting clones capable of expressing the recombinant protein. The presence and level of expression of the recombinant protein is controlled by SDS page electrophoresis under denaturing conditions. Escherichia coli BL21 (DE3) is used as the parent strain to create the producer strain. The advantage of using this strain as a basis for creating a producer strain is that BL21 (DE3) has the Lon OmpT phenotype, which eliminates the possibility of proteolytic cleavage of the synthesized His8-TrxL-Acip1 hybrid protein and contamination of the drug with the most active Escherichia coli proteases. The T7 RNA polymerase gene is integrated into BL21 (DE3) chromosomal DNA, which, together with the T7 promoter and T7 terminator in the pET-His8-TrxL-Acip1 plasmid, provides the production of His8-TrxL-Acip1 fusion protein by Escherichia coli cells by inducing isopropylthio-β-D -galactoside or lactose. Basal expression (until the inducer is added) of T7 RNA polymerase and His8-TrxL-Acip1 fusion protein is kept to a minimum due to the presence of a control system based on lac-operators and lac-repressor genes present in the pET-His8-TrxL-Acip1 plasmid , and in the chromosome of the producer strain.

The cells of the producer strain retain the cultural-morphological and physiological-biochemical characteristics of the parent strain of Escherichia coli. The cells are small, rod-shaped, gram-negative, 1 × 3.5 μm, motile, grow well on ordinary nutrient media (ΜΠΑ, MPB, LB-broth, LB-arap, minimal medium with glucose). Growth in liquid media is characterized by even turbidity; sediment easily sedimentes. Cells grow at temperatures from 4 ° C to 42 ° C with optimum pH from 6.8 to 7.5; as a source of nitrogen, both mineral ammonium salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract. When growing on a minimal medium, amino acids, glycerin, and carbohydrates are used as a carbon source. Cells show resistance to ampicillin (up to 500 μg / ml) due to the presence of the β-lactamase gene in plasmid pET-His8-TrxL-Acip1.

The producer strain is stored on dishes and jambs at a temperature of 4 ° C, transferring to fresh media once a month, as well as at a temperature of minus 70 ° C in LB medium with the addition of 10-30% glycerol.

The biosynthesis of the product (expression) is carried out as follows: the cells of the producer strain are grown in a nutrient medium (for example, LB, MBL or M9) with the addition of the necessary selection agent (100 μg / ml ampicillin) at a temperature of 20-37 ° C until the culture reaches an average or late logarithmic growth phase, stepwise increasing the volume of the culture fluid by successive transfers of material, after which synthesis of the hybrid protein is induced by the addition of isopropylthio-β-D-galactoside or lactose and incubated for an additional 2-24 h at a temperature of 20-37 ° C. An increase in the yield of the hybrid protein is achieved by forced aeration of the culture fluid and cultivation of the producer strain on enriched nutrient media (for example, with the addition of glucose, glycerol, dicarboxylic acids, amino acids, inorganic salts, including those containing trace elements).

Purification of the acipensin-1 antimicrobial peptide involves the following mandatory steps: cell disruption, metal chelate purification of the TrxL-Acip1 fusion protein, treatment of the hybrid protein with bromine cyan, separation of reaction products by repeated metal chelate purification, and purification of the target peptide by reverse phase HPLC.

To purify the cell material from impurities contained in the culture fluid, especially when fusion protein is isolated from a low density cell culture, it is desirable to pre-concentrate the cells by centrifugation or filtration. The destruction (lysis) of cells is carried out physically or chemically or by a combination of methods, for example, using an ultrasonic disintegrator, a French press, a Gaulin disintegrator, or using osmotic shock, detergents, chaotropic agents, hydrolytic enzymes (lysozyme, DNase). In order to reduce the load on the affinity sorbent, insoluble impurities from the cell lysate should be removed by centrifugation or filtration. Sorbent can be used for cleaning metal chelate containing chelating groups such as iminodiacetate (IDA), nitrilotriacetate (NTA) or karboksimetilaspartat (AGR, TALON) in a complex with cations Ni ^2+, Co ²⁺ or Cu ²⁺ [Chaga GS (2001 ) J Biochem Biophys Methods. 49 (1-3): 313-34]. Elution of the hybrid protein is carried out by decreasing the pH of the buffer solution or increasing the concentration of imidazole or adding EDTA to the buffer solution. The reaction with cyanogen bromide is carried out under standard conditions: the protein is dissolved in 60-90% trifluoroacetic, acetic or formic acid or in 6M guanidine chloride with the addition of 0.1 M hydrochloric acid, bromine is added in a 10-200-fold stoichiometric excess with respect to the number of residues methionine in the sample, incubated in the dark for 16-20 hours. After that, the products of the fusion protein cleavage reaction are purified by repeated metal chelate chromatography, thus separating the target peptide, carrier protein and uncleaved hybrid protein. Purification of acipensin-1 is carried out by reverse phase HPLC in an acetonitrile-water system with the addition of 0.1% TFA using an acetonitrile concentration gradient. The purity of the peptide can be increased by repeated purification by reverse phase HPLC.

Correspondence of the obtained preparation to natural acipensin-1 is established by methods of PAGE-electrophoresis under denaturing conditions, MALDI time-of-flight mass spectrometry, sequencing of the N-terminal amino acid sequence by the Edman method, testing of antimicrobial activity by the method of serial dilutions in a liquid nutrient medium. The degree of purification is determined by methods of PAGE-electrophoresis under denaturing conditions and MALDI-time-of-flight mass spectrometry, as well as using repeated reverse-phase HPLC.

In FIG. 1 is a physical map of a plasmid vector for expression of the antimicrobial peptide acipensin-1: BglII, XhoI — restriction sites; pBR322 Ori — plasmid replication initiation site; bla - gene for resistance to β-lactam antibiotics; lacI - lac-penpeccopa gene; T7 promoter - transcription promoter; T7 terminator - transcription terminator; lac operator - lac-penpeccopa binding site; RBS - ribosome binding site; His8-TrxL-Acip1 is a sequence encoding a fusion protein containing acipensin-1. In FIG. 2 shows an electrophoregram of a cell lysate containing His8-TrxL-Acip1 fusion protein: M — molecular weight standard; 1, 2 - total cellular protein before IPTG induction (0.1 and 0.2 p.u., respectively); 3, 4 - total cellular protein after IPTG induction (0.1 and 0.2 pu, respectively). In FIG. Figure 3 shows the chromatogram of purification of recombinant acipensin-1 by reverse phase HPLC (the arrow indicates the peak corresponding to the target peptide). In FIG. 4 shows the MALDI mass spectrum of acipensin-1 obtained by genetic engineering.

The invention is illustrated by examples.

Example 1

Construction of the plasmid vector pET-His8-TrxL-Acip1.

The nucleotide sequence of SEQ ID No. 2, containing a T7 RNA polymerase transcription promoter, a lac operator, a ribosome binding site and a hybrid polypeptide coding region (sequentially linked histidine octamer, thioredoxin with M37L substitution, bromine cyan and acipensin-1 cleavage site), are obtained by chemical-enzymatic synthesis using PCR The oligonucleotides used in PCR are synthesized by the solid-state phosphoamidite method with the growth of the oligonucleotide chain in the direction from the 3′-end to the 5′-end using protected phosphamidites - 5′-dimethoxytrityl-N-acyl-2′-deoxynucleoside-3′-O- (β-cyanoethyl diisopropylamino) phosphites activated with tetrazole.

The fragment encoding the carrier protein thioredoxin (M37L) was obtained by PCR amplification and directed mutagenesis using gene-specific primers using the pET32a (+) plasmid containing the thioredoxin gene as the initial template. The remaining sections of the pE-TrxL-Acip1 sequence are obtained by sequential annealing and elongation of mutually overlapping oligonucleotides, as well as annealing, elongation and amplification of intermediate synthesis products. At the final stage of the synthesis, the sequence is amplified using primers carrying restriction enzyme recognition sites BglII and XhoI at the 5 ′ ends. The amplification product is hydrolyzed with the indicated restriction enzymes, purified by electrophoresis in a 1.5% agarose gel, a 659 bp DNA band. isolated from the gel by electroelution in a 15% PEG-6000 solution and ligated with a 5.25 kbp DNA fragment obtained by treating the plasmid pET31b (+) with BglII and XhoI restriction enzymes. As a result of the ligase reaction, circular covalently closed DNA of 5912 bp is obtained. (Fig. 1). The ligase reaction products transform competent Escherichia coli DH10B cells prepared with 0.1 M calcium chloride. After transformation, the bacterial suspension is mixed with LB medium, grown for 1 h at 37 ° C and plated on Petri dishes with LB agar containing 50 μg / ml ampicillin.

The primary selection of clones containing the desired plasmid is carried out by the method of "PCR from clones" using primers on the plasmid backbone and insert. Selected clones are grown in a liquid nutrient medium and plasmid DNA is isolated, which is analyzed for the presence of insert using restriction analysis. The final structure of plasmids containing the desired fragment is confirmed by determining the nucleotide sequence using Sanger sequencing. According to sequencing data, a plasmid with an insert is selected whose nucleotide sequence is fully consistent with the planned one (SEQ ID No. 2).

Example 2

Obtaining a producer strain of Escherichia coli BL21 (DE3) / pET-His8-TrxL-Acip1 producing the His8-TrxL-Acip1 fusion protein and determining its productivity.

Competent Escherichia coli BL21 (DE3) cells prepared using 0.1 M calcium chloride were transformed with the plasmid pET-His8-TrxL-Acip1 described in Example 1. After the transformation, the bacterial suspension was mixed with LB medium and grown for 1 h at 37 ° C and plated on Petri dishes with LB agar containing 50 μg / ml ampicillin and 0.5% glucose. The plates are incubated at 37 ° C for 18 hours.

The grown colonies are transferred with a bacteriological loop in 10 ml of LB liquid medium containing 150 μg / ml ampicillin, grown to an optical density of OD ₆₀₀ 0.7 on a thermostatic shaker at a speed of 200 rpm ^-1 at 37 ° C, 0.3 are selected ml of culture for subsequent electrophoretic analysis, add isopropylthio-β-D-galactoside inductor to a concentration of 0.2 mM and continue incubation at 37 ° C for 5 h, taking 0.3 ml samples every hour to determine the optical density OD ₆₀₀ and subsequent elektroforeticheskog analysis. Equal aliquots of the cell suspension, taken before the inducer was introduced and after growth was complete, were centrifuged, the supernatant was separated, and the cell pellet was analyzed by PAGE by electrophoresis under denaturing conditions. For this, samples are lysed with a buffer containing 2% sodium dodecyl sulfate (SDS) in a water bath for 5 minutes. Electrophoresis is carried out in 15% SDS page in the presence of SDS. The gel is stained with 0.1% Coomassie G-250 in the presence of 25% isopropyl alcohol. The appearance of a distinct band in the region of 18.9 kDa in samples taken after induction indicates the ability of the strain to synthesize the His8-TrxL-Acip1 hybrid polypeptide (Fig. 2). A relative fusion protein content of about 30% is determined by scanning and densitometric analysis of the stained gels.

Example 3

Obtaining recombinant antimicrobial peptide acipensin-1.

Cells producing strain Escherichia coli BL21 (DE3) / pET -His8-TrxL-Acip1, obtained according to Example 2, were grown in LB liquid medium supplemented with 100 ug / ml ampicillin and 1% glucose until an optical density of culture OD ₆₀₀ 0.7 at a temperature of 37 ° C. Protein synthesis is induced using isopropyl-β-D-galactoside at a concentration of 1.0 mM, after which it is incubated for 4 hours at a temperature of 37 ° C. The level of expression of the hybrid protein, as well as the content of the hybrid protein, carrier protein and acipensin-1 in the preparation at the subsequent purification steps, is determined by SDS-PAGE electrophoresis in Tris-Tricin buffer system under denaturing conditions.

After fermentation, the cell biomass was separated by centrifugation at 4000 g for 10 min, resuspended in buffer A (50 mm Tris-HCl, 0.5 M NaCl, 20 mm imidazole, pH 7.8, 1 mm PMSF) and destroy cells using ultrasonic homogenizer. The resulting lysate is centrifuged (clarified) at 25,000 g for 20 minutes. All work on obtaining clarified lysate is carried out at a temperature of 4 ° C. Purification of the His8-TrxL-Acip1 fusion protein containing the octahystidine sequence as an affinity tag was carried out using metal chelate chromatography on a preparative column with Ni-NTA agarose equilibrated with buffer A. For this, the clarified lysate was applied to the column and the hybrid protein bound to the carrier was eluted with buffer In (50 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 7.8). Detection is carried out at a wavelength of 280 nm. The eluate containing the fusion protein is dialyzed relative to water for 16 hours through a 12 kDa membrane and freeze-dried.

The purified His8-TrxL-Acip1 fusion protein is dissolved in 80% trifluoroacetic acid at a concentration of 10 mg / ml, an equal mass of bromine cyan (1 g bromine cyan per 1 g protein) is added and kept at a temperature of 25 ° C in a dark place for 16 hours The reaction is stopped by adding five times the volume of deionized water, after which the sample is freeze-dried. The procedure for adding water and evaporation is repeated three times.

The reaction products are dissolved in 80% trifluoroacetic acid at a concentration of 5%. The insoluble precipitate was separated by centrifugation at 10,000 g for 10 min and discarded. The separation of the reaction products of the cleavage and purification of acipensin-1 is carried out using reverse-phase HPLC on preparative columns in the acetonitrile-water system with the addition of 0.1% TFA (Fig. 3). Detection is carried out at a wavelength of 214 nm. The fraction containing acipensin-1 is collected and freeze-dried. The output of the peptide in terms of 1 l of cell culture is 10 mg.

To determine the N-terminal amino acid sequence of acipensin-1, automatic microsequencing is used on a Procise 491 cLC Protein Sequencing System (PE Applied Biosystems). The identification of phenylthiohydantoin derivatives of amino acids is carried out on a 120A PTH Analyzer (PE Applied Biosystems). The method for the automatic determination of the amino acid sequence of peptides and proteins is based on the method of chemical degradation of the polypeptide chain according to the Edman method, which allows one to sequentially cleave the N-terminal amino acid residues in the form of phenylthiohydantoins and identify the cleaved amino acid derivatives by reverse-phase HPLC. As a result of automatic microsequencing, the amino acid sequences of the genetic engineering and natural acipensin-1 are identified.

To determine the molecular weight of the purified protein, a MALDI time-of-flight mass spectrometric analysis was used on a Reflex III instrument (Bruker Daltonics) equipped with a 336 nm UV laser. As a matrix, 2,5-dihydroxybenzoic acid is used in a mixture containing 20% acetonitrile and 0.1% trifluoroacetic acid. The resulting peak with m / z 5295.9 (Fig. 4) corresponds to the molecular ion of natural acipensin-1 (calculated molecular weight 5295.2).

Example 4

Testing the antimicrobial activity of recombinant acipensin-1.

The antimicrobial activity of recombinant acipensin-1 is determined by double serial dilutions of the peptide in LB / 2 liquid nutrient medium and incubation with test cultures (Staphylococcus aureus 209P and Escherichia coli C600).

Test cultures were seeded from canned food in 10 ml of LB liquid medium without antibiotic and grown for 16 hours at 37 ° C and 220 rpm ^-1 . An aliquot of the culture with a volume of 300 μl is added to 6 ml of LB medium and incubated on a rotary shaker at 37 ° C until the culture reaches an optical density of OD ₆₀₀ ~ 1.0. After that, the cells are pelleted by centrifugation, the culture is washed with 10 mM phosphate buffer (pH 7.4), and the cells are resuspended in the same buffer at a concentration of 2 · 10 ⁵ CFU / ml.

Testing is carried out in polystyrene 96-well plates. Aliquots of test cultures with a volume of 50 μl are added to 10 μl of solutions of pre-prepared dilutions of the test peptide. After adding culture, the plate is incubated for 2 hours at 37 ° C and vigorous stirring with a frequency of 1000 rpm ^-1 . Then add 60 μl of LB growth medium (to a total volume of 120 μl in the well) and continue incubation at 30 ° C and 1000 rpm ^-1 for 20 hours. The MIC value is determined visually as the minimum concentration of the peptide at which there is no growth of microorganisms .

The MIC value of acipensin-1, determined by the above method, with respect to Staphylococcus aureus 209P and Escherichia coli C600 is 0.6 ± 0.1 μM.

                         SEQUENCE LISTING

<110> IBCh RAS

       Ovchinnikova, Tatyana

       Balandin, Sergey

       Kokryakov, Vladimir

       Panteleev, Pavel

       Shamova, Olga

       Bolosov, Ilya

<120> PLASMID VECTOR pET-His8-TrxL-Acip1, Escherichia BACTERIA STRAIN

        coli FOR EXPRESSION OF ANTIMICROBE ACEPENSIN-1 PEPTIDE AND METHOD

        PREPARATION OF THE INDICATED PEPTIDE

<130> (Reference Number)

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 50

<212> PRT

<213> Acipenser gueldenstaedtii

<400> 1

Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr

1 5 10 15

Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg

            20 25 30

Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala Pro

        35 40 45

Val tyr

    fifty

<210> 2

<211> 665

<212> DNA

<213> Artificial Sequence

<220>

<223> Expression cassette

<220>

<221> BglII restriction site

<222> (1) .. (6)

<220>

<221> T7 promoter

<222> (30) .. (46)

<220>

<221> His8-TrxL-Acip1 coding sequence

<222> (117) .. (641)

<220>

<221> XhoI restriction site

<222> (660) .. (665)

<400> 2

agatctgcgg gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 60

ggataacaat tcccctctag agtcgacgga tcttacttta agaaggagat atacatatga 120

gccatcacca ccaccatcac catcacggat ctagcgataa aattattcac ctgactgacg 180

acagttttga cacggatgta ctcaaagcgg acggggcgat cctcgtcgat ttctgggcag 240

agtggtgcgg tccgtgcaaa ctgatcgccc cgattctgga tgaaatcgct gacgaatatc 300

agggcaaact gaccgttgca aaactgaaca tcgatcaaaa ccctggcact gcgccgaaat 360

atggcatccg tggtatcccg actctgctgc tgttcaaaaa cggtgaagtg gcggcaacca 420

aagtgggtgc actgtctaaa ggtcagttga aagagttcct cgacgctaac ctggccggat 480

ctgatccgat gtctggaaga ggaaagactg gcggtaaagc tcgtgctaag gcaaagactc 540

gctcctccag ggcaggactg cagttcccag tcggccgtgt ccacaggctg ctgcggaagg 600

gaaactatgc ccagcgtgtc ggcgctggag ccccggtcta ttaaggatcc gcgaattctc 660

tcgag 665

<210> 3

<211> 175

<212> PRT

<213> Artificial Sequence

<220>

<223> fusion protein

<400> 3

Met Ser His His His His His His His His His His Gly Ser Ser Asp Lys Ile

1 5 10 15

Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp

            20 25 30

Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys

        35 40 45

Leu Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys

    50 55 60

Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro

65 70 75 80

Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly

                85 90 95

Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys

            100 105 110

Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Asp Pro Met Ser Gly Arg

        115 120 125

Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr Arg Ser Ser

    130 135 140

Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg

145 150 155 160

Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala Pro Val Tyr

                165 170 175

Claims (3)

1. Plasmid vector pET-His8-TrxL-Acip1 for expression in Escherichia coli cells of the antimicrobial peptide acipensin-1 as part of the His8-TrxL-Acip1 fusion protein, consisting of two DNA fragments:
- BglII / XhoI fragment with the nucleotide sequence of SEQ ID No. 2, comprising a T7 RNA polymerase transcription promoter, a lac operator, a ribosome binding site and an affinity tag coding region, a thioredoxin protein and acipensin-1;
- BglII / XhoI fragment of plasmid pET31b (+), containing the T7 RNA polymerase transcription terminator, replication initiation site, β-lactamase gene, and lac-peppeccopa (lacI) gene. 2. The bacterial strain Escherichia coli BL21 (DE3) / pET-His8-TrxL-Acip1 is a producer of the His8-TrxL-Acip1 fusion protein obtained by transforming cells of the parent strain BL21 (DE3) with the pET-His8-TrxL-Acip1 plasmid vector according to claim one. 3. The method of producing the acipensin-1 antimicrobial peptide, comprising expressing the His8-TrxL-Acip1 fusion protein in the Escherichia coli BL21 (DE3) / pET-His8-TrxL-Acip1 producer strain of claim 2, cell lysis, His8 fusion protein purification -TrxL-Acip1 on a metal chelate carrier, cleavage of the His8-TrxL-Acip1 fusion protein with cyanogen bromide at the methionine residue introduced between the sequences of acipensin-1 and thioredoxin, repeated metal chelate purification and purification of the target peptide by reverse phase HPLC.

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