RU2456345C1 - RECOMBINANT PLASMID DNA pЕ-Trx-Lc-def, Escherichia coli STRAIN FOR EXPRESSION OF ANTIMICROBIAL PEPTIDE OF LENTIL DEFENSIN Lens culinaris AND METHOD FOR PRODUCING SAID PEPTIDE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention involves plasmid DNA pE-Trx-Lc-def for defensing expression in Escherichia coli cells as a part of the fusion protein Trx-Lc-def, transformation of the parental E. coli strain with said plasmid and further production of the target peptide from the producer strain.

EFFECT: increase of plant resistance to the infection in agriculture.

3 cl, 4 dwg, 1 tbl, 4 ex

Description

The invention relates to the field of biotechnology, in particular to the production of the antimicrobial peptide of the defensin of lentil ordinary Lens culinaris, which can be used as a medicine in medical and veterinary practice, as well as a tool that increases the resistance of plants to infection in agriculture.

Plant defensins are a class of cationic, cysteine-rich peptides with a length of 45-54 amino acid residues that play an important role in protecting plants from pathogenic microorganisms. Plant defensins are encoded by genes that form multi-gene families. Induction of the expression of these genes occurs under the influence of various biotic and abiotic factors. Plant defensins, like mammalian defensins, are multifunctional peptides and are characterized by a wide spectrum of in vitro biological activity. These peptides inhibit the growth of microorganisms and the proliferation of tumor cells, inhibit the activity of insect digestive enzymes, protein biosynthesis and HIV-1 reverse transcriptase, block ion channels, and inhibit the development of parasitic plants [Carvalho A. et al. (2009) Peptides. 30: 1007-1020].

Plant defensins exhibit antimicrobial activity in micromolar concentrations against a large number of phytopathogenic fungi and bacteria, and also have insecticidal activity and can be used in agriculture to create transgenic plants resistant to diseases. Traditional methods of combating pathogenic microorganisms, viruses and insect pests, based on the use of pesticides and insecticides, have such negative consequences as environmental pollution, the death of non-pathogenic microflora, insects and birds, as well as the emergence of resistant forms of pathogens and pests.

Plant defensins also inhibit the growth of some fungi pathogenic for humans, are characterized by a high specificity of action and the absence of toxic effects on mammalian cells. Recently, there has been an increase in the incidence of mycoses, the most often caused by pathogenic yeast-like fungi of the genus Candida and filamentous opportunistic fungi, such as Aspergillus, against which plant defensins are able to show their activity [Aerts A.M. et al. (2008) Cell. Mol. Life Sci. 65: 2069-2079]. The increase in the incidence of fungal infections is due to a change in the normal composition of human microflora, which occurs under the influence of various sensitizing factors and the unreasonable use of antiseptic agents and antibiotics. The appearance of pathogens of mycoses resistant to known antimycotics, an increase in the number of patients with immunodeficiency and the frequency of use of catheters and implants also lead to a change in the incidence rate of fungal infections and an increase in their clinical course. Due to the lack of antifungal vaccines, antimicrobials are often used to treat mycoses, often with a number of disadvantages. Used antimycotics, as a rule, require intravenous administration, interact with other drugs, are characterized by a narrow spectrum of antimicrobial activity, pronounced side effects and high toxicity [Thevissen K. et al. (2007) Drug Discov. Today. 12: 966-971].

Defensin (Lc-def) (SEQ ID No. 1), consisting of 47 amino acid residues and containing eight cysteine residues forming four disulfide bonds, was isolated from germinated seeds of lentil Lens culinaris. This peptide has antifungal activity and inhibits the growth of phytopathogenic fungi. The Lc-def precursor protein contains a signal peptide characteristic of plant defensins from 27 amino acid residues [Finkina E.I. et al. (2008) Biochem. Biophys. Res. Commun. 371 (4): 860-5].

Known closest to the claimed method for producing lentil defensin Lc-def, which consists in the homogenization of germinated lentil seeds, extraction, salting out of proteins and several successive purification steps, including gel filtration, ion exchange and reverse phase chromatography [Finkina E.I. et al. (2008) Biochem. Biophys. Res. Commun. 371 (4): 860-5]. The disadvantages of this method is the complex purification scheme and a low yield of 1 mg peptide per 1 kg of seeds.

The invention solves the problem of expanding the range of biologically active peptides and obtaining the antimicrobial peptide of the defensin of lentils ordinary Lens culinaris.

The problem is solved by:

1) recombinant plasmid DNA pE-Trx-Lc-def, consisting of two 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 sequence coding region, a carrier protein thioredoxin and lentils defensin;

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

2) a producer strain of the Trx-Lc-def fusion protein by transforming the Escherichia coli strain BL21 (DE3) with the indicated plasmid pE-Trx-Lc-def;

3) a method for producing lentil defensin, including culturing a producer strain of Escherichia coli BL21 (DE3) / pE-Trx-Lc-def, cell destruction, affinity purification of the Trx-Lc-def fusion protein on a metal chelate carrier, cleavage of the Trx-Lc- fusion protein def with bromine cyanide on the methionine residue introduced between the sequences of defensin and thioredoxin, and purification of the target peptide by reverse phase HPLC.

In order to prevent premature death of the producer microorganism during the expression of antimicrobial peptides in its cells and the associated loss of productivity, it is necessary to neutralize the toxicity of the synthesized product at this stage. The claimed plasmid DNA pE-Trx-Lc-def provides a high level of expression of defensin in E. coli due to the inclusion of its amino acid sequence in the Trx-Lc-def fusion protein (SEQ ID No. 3) carrying the sequence of thioredoxin A. Thioredoxin is able to accumulate in a high concentration (up to 40%) in the E. coli cytoplasm 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 defensin for a bacterial cell, but also to the possibility of its degradation in a heterologous system. To release the target peptide from the Trx-Lc-def fusion protein molecule, a methionine residue is introduced between the defensin and thioredoxin sequences, allowing selective cleavage of the fusion polypeptide with bromine cyan.

When splitting the Trx-Lc-def fusion protein with cyanogen bromide, it is advisable to use E. coli as a partner for heterologous expression of modified thioredoxin A, containing the substitution of the internal methionine residue for 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. 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 in the Trx-Lc-def fusion protein, which allows purification of the hybrid polypeptide by affinity chromatography on sorbents with immobilized (chelated) metal ions, i.e. metal chelate cleaning. As such a label, 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 [US Pat. No. 7176298].

The composition of the claimed plasmid DNA pE-Trx-Lc-def 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 lE repressor gene (lacI) is included in the plasmid pE-Trx-Lc-def. The claimed plasmid DNA includes the replication initiation site (Ori) of plasmid pBR322 and the β-lactamase marker gene, which determines the resistance of Escherichia coli cells transformed with plasmid pE-Trx-Lc-def to ampicillin and allows selection of cells containing plasmid DNA by growth on appropriate selective nutrient medium.

The construction of pE-Trx-Lc-def plasmid DNA expressing the Trx-Lc-def fusion protein containing the defensin sequence can be carried out by ligation of the BglII / XhoI fragment of the plasmid pET-31b (+) (Novagen) containing the replication initiation region, 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, defensin) 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. Ligase reaction products transform competent cells of the E. 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 with the insert is carried out using PCR and restriction analysis of the isolated plasmid DNA. The correct assembly of the construct is determined by plasmid DNA sequencing.

A producer strain of the Trx-Lc-def fusion protein containing the sequence of the defensin target peptide is obtained by transforming pE-Trx-Lc-def plasmid DNA with competent E. coli cells and selecting clones capable of expressing the recombinant protein. The presence and level of expression of a recombinant protein with a molecular weight of 19.1 kDa is monitored by SDS page electrophoresis under denaturing conditions. E. 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 excludes the possibility of proteolytic cleavage of the synthesized Trx-Lc-def fusion protein and contamination of the drug with the most active E. 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 pE-Trx-Lc-def plasmid, provides the production of the Trx-Lc-def fusion protein by E. coli cells by inducing isopropylthio-β- D-galactoside or lactose. Basal expression (until the inducer is added) of T7 RNA polymerase and Trx-Lc-def 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 plasmid pE-Trx-Lc-def , 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 E. coli strain. The cells are small, rod-shaped, gram-negative, 1 × 3.5 μm, motile, grow well on ordinary nutrient media (meat-peptone agar, meat-peptone broth, LB-broth, LB-agar, 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 are resistant to ampicillin (up to 500 μg / ml) due to the presence of the β-lactamase gene in plasmid pE-Trx-Lc-def.

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 3-24 hours 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 defensin antimicrobial peptide involves the following mandatory steps: cell disruption, metal chelate purification of the Trx-Lc-def fusion protein, treatment of the hybrid protein with bromine cyan, separation of reaction products by repeated metal chelate chromatography, 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, 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 can be removed by centrifugation or filtration. For metal chelate cleaning, a sorbent containing chelating groups such as iminodiacetate (IDA), nitrilotriacetate (NTA) or carboxymethylaspartate (CMA, TALON) in combination with Ni ²⁺ , Co ²⁺ or Cu ²⁺ cations can be used [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 or increasing the concentration of imidazole or adding EDTA to the buffer. 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 methionine residues in the sample, incubated in the dark for 16-20 hours. Next, the products of the fusion protein cleavage reaction are purified by repeated metal chelate chromatography, during which the carrier protein and the undigested hybrid protein bind to I eat the peptide goes breakthrough. Purification of defensin is carried out by reverse phase HPLC in an acetonitrile-water system with the addition of 0.1% trifluoroacetic acid using an acetonitrile concentration gradient. The purity of the peptide can be increased by repeated purification by reverse phase HPLC.

The identity of recombinant and natural defensin is established by methods of SDS page electrophoresis under denaturing conditions, MALDI time-of-flight mass spectrometry, sequencing of the N-terminal amino acid sequence by the Edman method, antimicrobial activity testing 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.

The invention is illustrated by graphic materials:

Figure 1. Physical map of recombinant plasmid DNA for expression of the defensin antimicrobial peptide: BglII, XhoI - restriction sites; pBR322 Ori — plasmid replication initiation site; bla - gene for resistance to β-lactam antibiotics; lacI - lac repressor gene; T7 promoter - transcription promoter; T7 terminator - transcription terminator; lac operator - lac repressor binding site; RBS - ribosome binding site; His8 is an affinity sequence of eight histidine residues; Trx is a sequence encoding thioredoxin; Lc-def is the sequence encoding defensin.

Figure 2. Electrophoretic analysis of the expression and purification of defensin: M - molecular weight standard; 1 - total cell lysate containing the Trx-Lc-def fusion protein; 2 - slip from the column with Ni sepharose; 3 - eluate containing a hybrid protein; 4 - dialysate; 5 - reaction products with bromine cyan; 6 - Lc-def purified by HPLC.

Figure 3. Chromatogram of purification of recombinant defensin by reverse phase HPLC.

Figure 4. MALDI mass spectrum of defensin obtained by genetic engineering.

The invention is illustrated by examples.

Example 1

Construction of recombinant plasmid DNA pE-Trx-Lc-def.

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 cleavage site and defensin), are obtained by PCR using enzymatic-chemical synthesis. 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-Trx-Lc-def 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 650 bp DNA strip. PEG-6000 is isolated from the gel by electroelution in a 15% solution and ligated with a 5.3 kbp DNA fragment obtained by treatment of plasmid pET31b (+) with BglII and XhoI restriction enzymes. As a result of the ligase reaction, circular covalently closed DNA of 5903 bp is obtained (figure 1). The ligase reaction products transform competent E. 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 for 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 by Sanger sequencing. According to sequencing data, a plasmid with an insert is selected, the nucleotide sequence of which is fully consistent with the planned one (SEQ ID No. 2).

Example 2

Obtaining a producer strain of Escherichia coli BL21 (DE3) / pE-Trx-Lc-def, producing a hybrid protein Trx-Lc-def, and determining its productivity.

Competent E. coli BL21 (DE3) cells prepared using 0.1 M calcium chloride were transformed with the plasmid pE-Trx-Lc-def 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 20 mmol / l glucose. The plates are incubated at 37 ° C for 18 hours.

The grown colonies are transferred with a microbiological 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 temperature-controlled shaker at a rotation speed of 200 min ^-1 at a temperature of 37 ° C, 0.3 ml of culture are selected for subsequent electrophoretic analysis, add the inducer isopropylthio-β-D-galactoside to a concentration of 0.2 mM and continue incubation at 30 ° C for 5 hours, taking 0.3 ml samples every hour to determine the optical density of OD ₆₀₀ and subsequent electrophoretic Alisa. 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 19 kDa in samples taken after induction indicates the ability of the strain to synthesize the Trx-Lc-def hybrid polypeptide (Fig. 2). The relative content of the hybrid protein is determined by scanning and densitometric analysis of stained gels.

Example 3

Obtaining a recombinant antimicrobial peptide defensin.

The cells of the producer strain Escherichia coli BL21 (DE3) / pE-Trx-Lc-def, obtained according to example 2, are grown in liquid nutrient medium LB with the addition of 100 μg / ml ampicillin and 20 mmol / l glucose until the culture reaches an optical density of OD ₆₀₀ 0.7 at a temperature of 37 ° C. The induction of protein synthesis is carried out using isopropyl-β-D-galactoside at a concentration of 0.2 mm, after which they are incubated for 4 hours at a temperature of 30 ° C. The expression level of the hybrid protein, as well as the content of the hybrid protein, the carrier protein and the target peptide in the preparation at the subsequent stages of purification, is determined using PAGE electrophoresis in Tris-glycine 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) containing 1 mm phenylmethyl sulfonyl fluoride (PMSF), and destroy cells using an 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 Trx-Lc-def fusion protein containing the octahytidine sequence as an affinity tag is carried out using metal chelate chromatography on a preparative Ni Sepharose column equilibrated with buffer A. For this, the clarified lysate is applied to the column, the column is washed with four times the volume of buffer A, and the bound with carrier hybrid protein buffer B (50 mm Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 7.8). The eluate containing the fusion protein is dialyzed against a 100-fold volume of water overnight at 4 ° C. through a 12 kDa membrane and freeze-dried.

The purified Trx-Lc-def fusion protein is dissolved in 80% trifluoroacetic acid at a concentration of 20 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.

Purification of the recombinant protein from the unsplit fusion protein and the carrier protein was carried out using repeated metal chelate chromatography in the same buffer system on a column of Ni Sepharose. Samples for application to a metal chelate column are prepared by dissolving a sample evaporated after bromine-cyan cleavage in buffer A at a concentration of 20 mg / ml. Residues of the acid are neutralized by adding an alkali solution (1 M NaOH) to a pH of 7.0.

The final purification of defensin is carried out using reverse-phase HPLC on a Reprosil-Pur C ₁₈ -AQ preparative column (d = 5 μm, 250 × 10 mm) in a linear gradient of 5-80% acetonitrile concentration for 60 min in the presence of 0.1% trifluoroacetic acid (figure 3). Detection is carried out at a wavelength of 214 nm. The fraction containing defensin is collected and freeze-dried. The yield of the peptide in terms of 1 l of cell culture is 3 mg.

Automatic microsequencing on a Procise 491 cLC Protein Sequencing System (PE Applied Biosystems) is used to determine the N-terminal amino acid sequence of defensin. 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 genetic engineering and natural defensin are identified.

To determine the molecular weight of the purified peptide, MALDI time-of-flight mass spectrometric analysis was performed 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 5441.29 (Fig. 4) corresponds to the molecular ion of natural defensin (calculated molecular weight is 5440.21 Da), stabilized by four disulfide bonds.

Example 4

Testing the antimicrobial activity of defensin.

The antimicrobial activity of recombinant Lc-def is determined by serial dilutions of the peptide in a liquid nutrient medium with a spore culture. Phytopathogenic fungi Aspergillus niger VKM F-2259, Aspergillus versicolor VKM F-114, Botrytis cinerea VKM F-85, Fusarium culmorum VKM F-844, Fusarium oxysporum TCXA-4 and Neurospora crassa VKM F-184 are grown in sterile potatoes on potatoes sucrose agar for 10 days at room temperature until active sporulation begins. The fungal mycelium grown on the surface is cut into 1 × 1 cm squares, placed in 3 ml of sterile water and incubated on a thermostatic shaker at 25 ° C in order to release and transfer the spores into suspension. The spore concentration is determined using a Goryaev camera. 110 μl of a spore suspension diluted in potato-sucrose broth to a concentration of 10 ⁴ spores / ml and 10 μl of sterile peptide solutions of various concentrations in 0.1% trifluoroacetic acid are added to the wells of a 96-well plate. Each test option is set in triplicate. The plate is incubated in a thermostatic shaker at 25 ° C for 48 hours. Spore germination is examined using a CX31 microscope (Olympus) and evaluated by measuring the absorbance of the culture in the wells at 620 nm using a Multiscan EX plate photometer (Thermo Scientific). As a negative control, 0.1% trifluoroacetic acid is used. The results are calculated using the program Multiscan Magic 3.1. For MIC ₅₀ (minimum inhibitory concentration) of defensin for this strain, the minimum concentration of the peptide is taken at which 50% inhibition of fungal growth is achieved compared to the negative control. The results of testing the activity of the recombinant peptide obtained by the claimed method are presented in table 1.

Table 1 Antifungal activity of defensin Phytopathogenic fungi MIC ₅₀ , μm / L Aspergillus niger VKM F-2259 28.7 Aspergillus versicolor VKM F-114 18.5 Botrytis cinerea VKM F-85 9.25 Fusarium culmorum F-844 18.5-37 Fusarium oxysporum TCXA-4 > 37 Neurospora crassa VKM F-184 9.25-18.5

The resulting recombinant Lc-def inhibits the growth of the phytopathogenic fungus A. niger, which causes the development of aspergillic rot in plants and aspergillosis in humans, with the same efficiency as natural defensin. The recombinant peptide also inhibits the growth of five other phytopathogenic fungi, namely A. versicolor, F. culmorum, F. oxysporum, N. crassa and B. cinerea. The most sensitive to recombinant Lc-def is the culture of N. crassa, the growth inhibition efficiency of which after 48 hours is 100% for defensin at a concentration of 37 μM / L.

Claims (3)

1. Recombinant plasmid DNA pE-Trx-Lc-def for expression in Escherichia coli cells of the antimicrobial lentil defensin peptide Lens culinaris as part of the Trx-Lc-def fusion protein, consisting of two DNA fragments ligated at the sticky ends:
- 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 defensin;
- 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) / pE-Trx-Lc-def is a producer of the Trx-Lc-def fusion protein obtained by transforming the cells of the parental BL21 (DE3) strain of plasmid DNA pE-Trx-Lc-def. 3. A method of obtaining a defensin antimicrobial peptide, comprising expressing a Trx-Lc-def fusion protein in an Escherichia coli BL21 (DE3) / pE-Trx-Lc-def producer strain, cell lysis, affinity purification of a Trx-Lc-def fusion protein on a metal chelate media, cleavage of the Trx-Lc-def fusion protein with cyanogen bromide according to the methionine residue introduced between the defensin and thioredoxin sequences, repeated metal chelate purification and purification of the target peptide by reverse phase HPLC.

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