RU2415940C1 - PLASMID VECTOR pE-Lc-LTP, Escherichia coli BACTERIUM STRAIN FOR EXPRESSION OF LIPID-TRANSPORTING LENTIL PROTEINS Lens culinaris AND METHOD FOR PRODUCING SAID PROTEINS - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: described is a plasmid vector pE-Lc-LTP for protein Lc-LTP expression in Escherichia coli cells, containing a gene of the hybrid polypeptide Trx-Lc-LTP including the amino acid sequence Lc-LTP, a sequence of the protein thioredoxin and an affine mark. Presented is a producer strain of said hybrid polypeptide based on the E.coli bacteria strain. Offered is a method for producing the end protein Lc-LTP by expression of the hybrid polypeptide Trx-Lc-LTP containing it in the cells of said strain, cell destruction, affine purification of the hybrid polypeptide Trx-Lc-LTP on a metalchelate carrier, hybrid protein breakdown in a site located between the sequences Lc-LTP and thioredoxin, and purification of the end protein Lc-LTP by reverse phase HPLC. The invention can find application both in agriculture as factors improving plant resistance to infections, and in medical practice for food allergy diagnostics and treatment.

EFFECT: enhanced efficiency and simplification of the technology of lipid-transporting lentil proteins Lens culinaris.

9 cl, 4 dwg, 6 ex

Description

The invention relates to the field of biotechnology, in particular to the production of lipid-transporting proteins of lentils Lens culinaris, which can be used in agriculture as factors that increase the resistance of plants to infections, and in medical practice for the diagnosis and treatment of food allergies.

Plant lipid-transporting proteins (LTPs) are a family of small cationic proteins with a molecular weight of about 7 or 9 kDa. These proteins are characterized by high stability, they are resistant to chemical and thermal denaturation, as well as to the action of proteases. The spatial structure of LTP is characterized by the presence of a hydrophobic depression formed by three or four α-helices and a long C-terminal fragment. It has been established that LTP is involved in many biological processes in plants, such as synthesis of cutane, embryogenesis, adaptation to stressful environmental conditions, and protection of plants from phytopathogens.

A characteristic property of plant LTP is the ability to bind and transfer lipids through biomembranes in vitro. Plant LTPs bind C ₁₀ -C ₁₈ chain fatty acids, acyl coenzyme A derivatives, phospholipids, skin lipids, galactolipids, prostaglandin B ₂ and amphotericin B. These proteins also carry phospholipids through membranes. These properties of plant-based LTPs make it possible to use them as ligand-binding proteins to create targeted drug and cosmetic delivery systems.

Plant LTPs have antimicrobial activity against both gram-positive and gram-negative bacteria, and also inhibit the growth of a wide range of phytopathogenic fungi. These properties have been successfully used to create transgenic plants that carry plant LTP genes and are highly resistant to disease.

Representatives of the LTP family are plant allergens involved in the development of allergic reactions to pollen, plant products and latex. Food allergens are LTP isolated from a number of fruits (apple, peach, plum, cherry, orange, grapes, etc.), vegetables (lettuce, asparagus, onions, carrots), nuts (hazelnuts, walnuts, chestnuts) and cereals (corn, barley). Pollen allergens are LTP from olives, celery, arabidopsis, Chernobyl, turnipsis and others. Recently, the relevance of obtaining recombinant protein allergens, which are biochemically and immunologically well characterized and can be used to diagnose and treat allergies, has been increasing.

Lens culinaris lentil lipid-transporting proteins (Lc-LTP) (SEQ ID Nos. 1-8) comprise a subfamily of lipid-transporting proteins isolated from germinated lentil seeds of ordinary Lens culinaris and inhibiting the growth of the soil phytopathogenic bacterium Agrobacterium tumefaciens [Finkin E. I. et al. (2007) Biochemistry, 72 (4): 533-43]. These proteins are composed of 92-93 amino acid residues and contain eight cysteine residues forming four disulfide bonds. Lc-LTP 1-8 precursor proteins contain a typical plant LTP signal peptide of 24-25 residues.

A known method of producing Lc-LTP, 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. (2007) Biochemistry, 72 (4): 533-43]. The disadvantages of this method is the complex purification scheme and the difficulty of purification of the target protein from impurities of related isoforms.

The invention solves the problem of expanding the range of biologically active vegetable proteins and obtaining lipid-transporting proteins of lentils Lens culinaris (Lc-LTP).

The problem is solved by constructing the plasmid vector pE-Lc-LTP for expression of Lc-LTP proteins in Escherichia coli cells containing the Trx-Lc-LTP hybrid polypeptide gene, including the amino acid sequence Lc-LTP, the thioredoxin protein sequence and affinity tag, creating a strain producer of this hybrid polypeptide based on the E. coli strain by transforming it with the indicated vector and a method for producing the target Lc-LTP protein by expressing the Trx-Lc-LTP hybrid polypeptide containing it in cells of the specified strain, sheniya cells, affinity purification of the fusion polypeptide Trx-Lc-LTP on the metal chelate carrier, the fusion protein cleavage site located between the sequences of Lc-LTP and thioredoxin and purification Lc-LTP target protein by reverse-phase HPLC.

A feature of the proposed vector pE-Lc-LTP is the presence in its composition of a gene encoding one of the proteins of the subfamily of lipid-transporting proteins of lentils (Lc-LTP). The high level of expression and solubility of Lc-LTP in E. coli cells is achieved due to the fact that this gene encodes a Trx-Lc-LTP fusion protein containing, along with the amino acid sequence of the Lc-LTP protein, a thioredoxin protein as a carrier protein. In order to further release the target Lc-LTP protein from the Trx-Lc-LTP fusion protein molecule, at least one site is introduced between the Lc-LTP and thioredoxin sequences, allowing the chemical polypeptide to be selectively cleaved by a chemical or enzymatic method. The chemical cleavage site may be a Met residue (cyanogen bromide cleavage), Asp-Pro dipeptide (acid hydrolysis digestion when heated), Asn-Gly dipeptide (hydroxylamine cleavage). The enzymatic cleavage site may be a thrombin, enterokinase, factor Xa, PreScission protease, TEV protease or HRV 3C protease cleavage site [Amau J. et al. (2006) Protein Expr Purif. 48 (1): 1-13].

When splitting the Trx-Lc-LTP fusion protein with cyanogen bromide, it is advisable to use E. coli modified thioredoxin A as a partner for heterologous expression, 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. In a preferred case, the cleavage of the hybrid protein gives only one by-product, significantly different in physicochemical properties from the target protein, which facilitates the process of purification of the latter.

Purification of the target Lc-LTP protein is simplified by the inclusion of an affinity tag, an amino acid sequence that allows affinity purification of the hybrid polypeptide on sorbents with immobilized (chelated) metal ions, to the Trx-Lc-LTP hybrid polypeptide. 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 into the region separating the carrier protein and the target protein [Terpe K. (2003) Appl Microbiol Biotechnol. 60 (5): 523-33]. HAT and 6 × HN fragments are also used as an affinity tag for metal chelate purification [U.S. Pat. No. 7,176,298].

The composition of the proposed vector pE-Lc-LTP includes regulatory elements that control the expression of a hybrid protein: a promoter for RNA polymerase, a bacterial ribosome binding site, start and stop codons. As a promoter, a natural (lac, T5, T7) or artificial (tac) transcription initiation site is used. To suppress the basal expression of the hybrid protein gene, at least one lac repressor binding site (lac operator) can be introduced into its regulatory region, and an additional lac repressor gene (lacI) can be introduced into the vector.

The composition of the proposed vector pE-Lc-LTP also includes regulatory elements necessary for its replication in E. coli cells, for example, ori (origin of replication) of the plasmid pBR322, pUC19 or pSC101.

The proposed vector contains at least one marker gene, allowing selection of cells containing vector DNA by growing on an appropriate selective nutrient medium. The marker gene can be a gene that determines resistance to an antibiotic or family of antibiotics, for example, ampicillin (bla), tetracycline (tet), kanamycin (kan), streptomycin (str) or chloramphenicol (cat).

The construction of a new vector pE-Lc-LTP vector expressing the Trx-Lc-LTP fusion protein containing the sequence of the target Lc-LTP protein can be performed by ligation of a plasmid fragment containing the replication initiation region and marker gene with an insert encoding the fusion protein gene . An artificial gene that encodes a hybrid 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, terminator, operator, ribosome binding site, thioredoxin, affinity sequence, Lc-LTP) is based on data on their nucleotide sequences available from open sources (GenBank, EMBL-Bank, DDBJ ) The template for amplification of the thioredoxin sequence is the bacterial genome, or plasmid pET-32a (+). 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 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 vector with the insert is carried out using PCR, as well as restriction analysis of the isolated vector DNA. The correct assembly of the construct is determined by vector DNA sequencing.

The Trx-Lc-LTP fusion protein producing strain containing the sequence of the target Lc-LTP protein is obtained by transforming competent E. coli cells with pE-Lc-LTP vector DNA and selecting clones capable of expressing the recombinant protein. The presence and level of expression of the recombinant protein is controlled using denaturing PAAG electrophoresis. As a parent strain to create a producer strain, an E. coli strain is used that is capable of carrying out controlled (inducible) transcription and translation of the hybrid protein gene in pE-Lc-LTP. For example, a producer strain for expression of a hybrid protein under the control of promoters for E. coli RNA polymerase (lac, tac, T5) in combination with a lac operator can be obtained on the basis of a strain that provides a high level of expression of lac-penpeccopa (M15 ( pREP4), XL I-Blue, JM109). When using the T7 bacteriophage promoter, it is necessary to use a strain containing the T7 RNA polymerase gene under the control of the lacUV5 promoter, for example, BL21 (DE3) or BL21 (DE3) Origami, or to infect the strain with CE6 helper phage containing the T7 RNA polymerase gene.

The cells of the producer strain retain the cultural-morphological and physiological-biochemical characteristics of the parent E. coli strain. Small rod-shaped cells, gram-negative, 1 × 3.5 μm, motile, grow well on ordinary nutrient media (MPA, MPB, 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 of the amino acid, peptone, tryptone, yeast extract are used. When growing on a minimal medium, amino acids, glycerin, and carbohydrates are used as a carbon source.

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 selective agents (for example, 100 μg / ml ampicillin) at a temperature of 20-37 ° C until the culture middle or late logarithmic growth phase, stepwise increasing the volume of the culture fluid by successive transfers of material, after which protein synthesis is induced and incubated for an additional 2-24 hours at a temperature of 20-37 ° C. An increase in the yield of the recombinant 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 lipid-transporting protein Lc-LTP includes the following mandatory steps: cell disruption, metal chelate purification of the Trx-Lc-LTP fusion protein, treatment of the fusion protein with a reagent that cleaves it at a site inserted between the thioredoxin and target protein sequences, separation of reaction products and purification of the target Lc-LTP protein by reverse phase HPLC.

To purify cell material from impurities contained in the culture fluid, especially when isolating protein from a cell culture with a low density, 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. French press, Gaulin disintegrator, using osmotic shock, detergents, chaotropic agents, hydrolytic enzymes. 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 [Chaga G S. ( 2001) J Biochem Biophys Methods. 49 (1-3): 313-34]. Elution of the hybrid protein is carried out using a pH gradient or imidazole concentration, or by adding to the EDTA buffer. If necessary, purification of the products of the fusion protein cleavage reaction is carried out using repeated metal chelate chromatography. At the final stage, repeated purification of the target protein by reverse-phase HPLC can be carried out.

The identity of the obtained Lc-LTP to a natural protein is established by methods of denaturing electrophoresis in SDS page, MALDI time-of-flight mass spectrometry, sequencing of the N-terminal amino acid sequence by the Edman method, testing of antimicrobial activity by serial dilution in a liquid nutrient medium. The degree of purification is determined by the methods of denaturing PAGE-electrophoresis and MALDI time-of-flight mass spectrometry, as well as by means of repeated reverse-phase HPLC.

Figure 1 presents a physical map of a recombinant vector for the expression of Lc-LTP. Figure 2 shows the results of electrophoretic analysis of the hybrid protein and Lc-LTP at different stages of purification. Figure 3 presents the chromatogram of the purification of Lc-LTP by reverse phase HPLC. Figure 4 shows the mass spectrum of purified Lc-LTP2.

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA pE-Lc-LTP for the expression of Lc-LTP

The nucleotide sequence (SEQ ID No. 9, nucleotides 10-807) containing the T7 RNA polymerase transcription promoter, a lac operator, a ribosome binding site, and a hybrid polypeptide coding region containing a sequentially linked histidine octamer, thioredoxin with M37L substitution, cleavage site by acid hydrolysis, a bromine cyan cleavage site and Lc-LTP, are obtained by enzyme-chemical synthesis using polymerase chain reaction (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 fragment encoding mature Lc-LTP was obtained by PCR using the plasmid pGEM-T (Promega) with the cloned cDNA fragment of the precursor of the Lc-LTP2 isoform as a template. To obtain this plasmid, total RNA is isolated from germinated lentil seeds of Lens culinaris, a reverse transcription reaction is performed with an oligo-dT primer, and then the 3'-ends of Lc-LTP cDNA are rapidly amplified using degenerate gene-specific primers to a conserved region corresponding to the signal the Lc-LTP peptide, then the resulting amplicon is cloned into the pGEM-T vector, the clones are screened and a clone containing the desired plasmid is found using Sanger sequencing.

The remaining sections of the pE-Lc-LTP 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 by the indicated restriction enzymes, purified by electrophoresis in a 1.5% agarose gel, a 790 bp DNA band. PEG-6000 is isolated from the gel by electroelution in a 15% solution and ligated with a 5.3 thousand bp DNA fragment obtained by processing the plasmid pET31b (+) with BglII and XhoI restriction enzymes. As a result of the ligase reaction, circular covalently closed DNA of 6041 bp is formed. 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, the plasmid is selected in which the nucleotide sequence of the ligated insert is completely identical to the planned one (FIG. 2, SEQ ID No. 9).

Example 2

Construction of recombinant plasmid DNA pE-Lc-LTP for the expression of Lc-LTP

DNA fragment (SEQ ID No. 10, nuquotides 10-784) containing the T7 RNA polymerase transcription promoter, a ribosome binding site and a hybrid polypeptide coding region containing the sequentially linked histadine octamer, thioredoxin (M37L), acid digestion site, site cleavage with cyanogen bromide and Lc-LTP is obtained by enzyme-chemical synthesis as described in Example 1, using the plasmid pGEM-T with the cloned cDNA fragment of the Lc-LTP4 isoform as an amplification matrix for the fragment encoding Lc-LTP. The amplification product is hydrolyzed with restriction enzymes BglII and XhoI, purified by electrophoresis in a 1.5% agarose gel, a 765 bp DNA band. PEG-6000 is isolated from the gel by electroelution in a 15% solution and ligated with a 3.5 kb DNA fragment obtained by treatment of plasmid pET20b (+) with restriction enzymes BglII and XhoI. As a result of the ligase reaction, a ring covalently closed DNA of 4270 bp is formed. Further operations are performed as described in Example 1. Using the Sanger sequencing method, the nucleotide sequence of the obtained construct is confirmed to be consistent with SEQ ID No. 10.

Example 3

Construction of recombinant plasmid DNA pE-Lc-LTP for the expression of Lc-LTP

A DNA fragment (SEQ ID No. 11, nucleotides 119-743) encoding a hybrid polypeptide containing sequentially linked E. coli thioredoxin A, an acid hydrolysis cleavage site, a bromine cyan cleavage site and Lc-LTP, is obtained by enzymatic-chemical synthesis as described in Example 1, using the plasmid pGEM-T with the cloned cDNA fragment of the Lc-LTP8 isoform as the matrix for amplification of the fragment encoding Lc-LTP. The amplification product is hydrolyzed with restriction enzymes BamHI and BglII, purified by electrophoresis in a 1.5% agarose gel, a 615 bp DNA band. isolated from the gel by electroelution in a 15% solution of PEG-6000 and ligated with linearized plasmid DNA pQE-70 (Qiagen) of 3.4 thousand bp obtained by treatment with restriction enzymes BamHI and BglII. As a result of the ligase reaction, a ring covalently closed DNA of 4035 bp is formed. Further operations are performed as described in Example 1. Using the Sanger sequencing method, the nucleotide sequence of the obtained construct is confirmed to be consistent with SEQ ID No. eleven.

Example 4

Obtaining a producer strain of E. coli producing a hybrid protein containing Lc-LTP, and determining its productivity

The E. coli BL21 (DE3) cells are transformed with the plasmid pE-Lc-LTP described in Example 1. 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 with a rotation speed of 200 min ^-1 at a temperature of 37 ° C, 0.3 ml of culture is taken for subsequent electrophoretic analysis, an inducer of isopropylthio-β-galactoside is added to a concentration of 0.2 mM and incubation is continued at a temperature of 37 ° C in for 5 hours, taking 0.3 ml samples every hour determination of optical density OD ₆₀₀ and subsequent electrophoretic 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 SDS-PAGE under denaturing conditions. For this, the samples are lysed with a buffer containing 2% SDS in a water bath for 5 minutes. Electrophoresis is carried out in 15% SDS-PAGE. The gel is stained with 0.1% Coomassie G-250 in the presence of 25% isopropyl alcohol according to standard methods. The appearance of a distinct band at 23 kDa in the samples taken after induction indicates the ability of the strain to synthesize a hybrid polypeptide containing Lc-LTP. A relative recombinant protein content of about 20% is determined by scanning and densitometric analysis of the stained gels.

Example 5

Obtaining recombinant lipid-transporting protein Lc-LTP

The cultivation of cells of the producer strain of Eschenchia coli obtained according to example 4, is carried out as follows:

The producer strain cells are grown in LB liquid medium with the addition of 100 μg / ml ampicillin and 1% glucose until the culture reaches an optical density of OD ₆₀₀ 0.7 at 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 and Lc-LTP2 in the preparation at different stages of purification, is determined using PAGE electrophoresis under denaturing conditions (Fig. 2).

After fermentation, the cells of the hybrid protein producer (biomass) are 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 destroyed using an ultrasonic homogenizer. The resulting lysate is centrifuged 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 fusion protein containing the affinity octahystidine sequence is carried out using metal chelate chromatography on a preparative column with Ni-NTA agarose equilibrated with the same buffer A. For this, the clarified lysate is applied to the column and the fusion protein bound to the carrier is eluted with buffer B (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 hybrid protein is dialyzed relative to water overnight and freeze-dried.

The purified hybrid protein is dissolved in 80 mg 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 25 ° C in a dark place for 16 hours. The reaction is stopped by adding five times 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. Insoluble precipitate was separated by centrifugation and discarded. Purification of Lc-LTP2 is carried out using reverse phase HPLC on preparative columns in a linear gradient of the concentration of acetonitrile 5-80% for 60 min in the presence of 0.1% TFU (figure 3). Detection is carried out at a wavelength of 214 nm. The fraction containing Lc-LTP2 is collected and freeze-dried.

To determine the N-terminal amino acid sequence of Lc-LTP2, automatic microsequencing is used on a Precise 491 cLC Protein Sequencing System (PE Applied Biosystems). The identification of phenylthiohydantoin derivatives of amino acids is carried out on a 120A PTH Analyzer (Applied Biosystems). The methodology 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 sequentially cleaving N-terminal amino acid residues in the form of phenylthiohydantoins and identifying cleaved amino acid derivatives by reverse phase HPLC. As a result of automatic microsequencing, the amino acid sequences of the genetic engineering and natural Lc-LTP2 were identified.

To determine the molecular weight of the purified protein, 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 t / g 9283,416 (Fig. 4) corresponds to the molecular ion of natural Lc-LTP2 (the molecular weight of the tracer is 9282.73 Da).

Example 6

Antimicrobial Activity Testing Lc-LTP

The antibacterial activity of natural and recombinant Lc-LTP2 against Agrobacterium tumefaciens strain A281 is determined by serial dilution in a liquid nutrient medium using 96-well plates. Bacterial cells were cultured overnight at 30 ° C in a sterile Petri dish with LB agar. Fresh biomass of the test culture is introduced into 3 ml of doubly diluted LB medium (ЅLB), and incubated at 30 ° C on a rotary shaker until the cells reach 1 ÷ 1.5 optical density units, which corresponds to 2 · 10 ⁸ cells / ml. Aliquots (110 μl) of a tenfold diluted exponential culture in LB medium are supplemented with 10 μl of sterile protein solutions of various concentrations in 0.1% TFA and incubated in a thermostatic shaker at 30 ° C. Bacterial growth was evaluated microspectrophotometrically by measuring the absorbance of the culture in the wells at 620 nm using a Multiscan EX plate photometer (Thermo). As a negative control using 0.1% TFU. The resulting recombinant Lc-LTP2 inhibits the growth of gram-negative phytopathogenic bacteria Agrobacterium tumefaciens with the same efficiency as the natural protein. In the presence of genetically engineered or natural Lc-LTP2 at concentrations of 40.5 μM, after 24 hours, 50% inhibition of bacterial cell growth is observed.

Claims (9)

1. Plasmid vector pE-Lc-LTP for expression of Lens culinaris lentil lipid transporting proteins in Escherichia coli cells, containing the Trx-Lc-LTP hybrid polypeptide gene comprising the thioredoxin protein amino acid sequence, affinity tag, cleavage site, and lipid transporting protein sequence Lc-LTP selected from the sequence group SEQ ID Nos. 1-8, where
affinity tag - an amino acid sequence that allows affinity purification of a hybrid polypeptide on sorbents with immobilized metal ions,
cleavage site - a portion of the amino acid sequence located between the sequences of the lipid-transporting protein Lc-LTP and thioredoxin, allowing partial cleavage of the hybrid polypeptide by chemical or enzymatic methods. 2. The plasmid vector according to claim 1, containing the T5 promoter in the composition of the hybrid polypeptide gene. 3. The plasmid vector according to claim 1, containing the T7 promoter in the composition of the hybrid polypeptide gene. 4. The plasmid vector according to claim 1, wherein the thioredoxin protein sequence is an Escherichia coli thioredoxin A sequence with replacement of the internal Met residue by Leu, and the cleavage site is the Met residue. 5. The plasmid vector according to claim 1 with a nucleotide sequence selected from the group of sequences SEQ ID Nos.9-11. 6. The bacterial strain Escherichia coli is a producer of the Trx-Lc-LTP hybrid polypeptide obtained by transforming cells of the parent strain BL21 (DE3) or XLl-Blue with the plasmid vector pE-Lc-LTP. 7. A method for producing Lens culinaris lentil lipid-transporting proteins, comprising expressing the target protein in the Trx-Lc-LTP fusion polypeptide in a producer strain obtained by transforming cells of the parent strain of the bacterium Escherichia coli BL21 (DE3) with the plasmid vector pE-Lc-LTP cell lysis, affinity purification of the fusion protein on a metal chelate carrier, cleavage of the fusion protein at a site located between the Lc-LTP and thioredoxin sequences, and purification of the target lipid-transporting protein by reverse-phase HPLC. 8. The method according to claim 7, where the fusion protein is cleaved with cyanogen bromide in an acidic medium at the remainder of Met. 9. The method according to claim 7, where the fusion protein is cleaved by acid hydrolysis via Asp-Pro bond.

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