RU2316595C1 - Method for preparing antimicrobial peptide arenicin - Google Patents

Abstract

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing peptide arenicin possessing the antimicrobial activity. Method involves culturing the recombinant strain-producer Escherichia coli BL21(DE3)/pE-His8-TrxL-Ar2 in nutrient medium followed by disruption of bacterial cells, isolation of inclusion bodies comprising fusion protein His8-TrxL-Ar2. Then this fusion protein is purified by the method of metal-chelating chromatography, subjected for solubilization and the following splitting with cyanogen bromide by methionine residue in acid medium. Then reaction products are separated and arenicin is subjected for final purification by reversed-phase HPLC method. Using the invention provides expanding assortment of antimicrobial preparations of broad spectrum of effect and to carry out the high-technological effectiveness of production of arenicin. Prepared peptide is similar to peptide isolated from annelid worm Arenicola marina and can be used in medicinal and veterinary practice as antibiotic of the broad spectrum of effect.

EFFECT: improved preparing method of peptide.

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Description

The invention relates to the field of biotechnology, and in particular to the production of genetically engineered arenicin, an antimicrobial peptide of the annelid worm Arenicola marina, which can be used in medical and veterinary practice as a broad-spectrum antibiotic.

Arenicin is an endogenous antibiotic of the invertebrate Arenicola marina (marine peskozhil), belonging to the class Polychaeta (polychaete) type Annelida (annelids) [US Pat. RF №2261866, С07К 14/435, А61Р 31/04, 2005]. The arenicin molecule is an unmodified peptide chain containing 21 amino acid residues and stabilized by one disulfide bond, closing the cycle of 18 amino acid residues. Arenicin is produced by coelomocytes (phagocytic cells of the worm's coelom) and has antibacterial and antifungal activity. The mechanism of action of arenicin presumably consists in the selective violation of the barrier function of cell membranes of pathogenic microorganisms, which does not affect the cell membranes of the host organism. Arenicin is a representative of a fundamentally new family of antimicrobial peptides and can be used as a broad-spectrum antibiotic.

Currently, clinical medicine is faced with the problem of resistance of pathogens to antibiotics from the existing arsenal of drugs, which in some cases no longer provide effective control over the pathogen in the development of chronic and recurrent infectious diseases. Antimicrobial peptides are the leading molecular factors of the innate immunity system of eukaryotic organisms, without which it is impossible to survive in an environment abundant with potential pathogens. Antimicrobial peptides with a wide spectrum of biological action are associated with one of the evolutionarily oldest mechanisms of emergency protection of animals and humans from infection. Currently shown immunomodulating activity of antimicrobial peptides [Kamysz W., Okroj M., Lukasiak J. // Novel properties of antimicrobial peptides. Acta Biochim. Pol., 2003, v. 52 (2), p. 461-469]. Given the presence of pronounced antibiotic and immunomodulating activity in natural antimicrobial peptides and their special role in the functioning of the innate immunity system [Papagianni M. // Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications. Biotechnology Advances, 2003, v.21, p.465-499], many foreign pharmaceutical and biotechnological companies have begun to create a new class of antibiotics based on them. Peptide antibiotics are currently undergoing clinical trials in technologically developed countries, which are recommended as therapeutic agents in the treatment of sepsis, meningitis, pneumonia, pulmonary infections in cystic fibrosis (cystic fibrosis), gastric ulcer, diabetic foot infections, and also in the treatment of candidiasis of the mucous membrane oral cavity, mucositis, gingivitis, acne, impetigo, mycosis, when healing burns and wounds complicated by a secondary infection, etc. [Mcphee J.B., Hancock R.E.W. // Function and therapeutic potential of host defense peptides. J. Peptide Science. - 2005, vol. 11, p. 677-687].

A known method for producing the antimicrobial peptide of indolicidin, including culturing the E. coli strain BL21 (DE3) transformed with the plasmid pET32a: Ind3, chemical lysis of cells in a buffer containing 6 M guanidine chloride, purification of the hybrid protein using metal chelate chromatography, cleavage with bromocyanine acidic medium according to methionine residues, separation of reaction products and purification of indolicidin by reverse phase HPLC [Morin KM, Arcidiacono S., Beckwitt R., Mello CM // Recombinant expression of indolicidin concatamers in Escherichia coli. Appl. Microbiol. Biotechnol., 2006, v. 70, p.698-704]. The main disadvantage of this method is the low yield of the recombinant peptide of 150 μg with 1 l of cell culture.

Known closest to the claimed method for producing the antimicrobial peptide of adenoregulin, including culturing the producer strain E. coli BL21 (DE3) / pET32a-adr, destroying bacterial cells by disintegration, separating the soluble fraction of the cell protein by centrifugation, purification of the hybrid protein using metal chelate chromatography, splitting with bromine cyan in an acidic medium according to methionine residues, separation of the reaction products and final purification of adenoregulin by reverse phase and exclusion HPLC [Zhou YX, Cao W., Luo Q.P., Ma Y.S., Wang J.Z., Wei D.Z. // Production and purification of a novel antibiotic peptide, adenoregulin, from a recombinant Escherichia coli. Biotechnology Letters, 2005, v. 27 (10), p. 725-730]. The disadvantage of the system is the difficulties encountered during chromatographic purification of the target peptide after cleavage of the hybrid protein with cyanogen bromide. The original plasmid pET32a encodes a methionine residue in the natural sequence of thioredoxin A and four methionine residues in the linker that separates thioredoxin A from the sequence of the target polypeptide. As a result, when bromide is cleaved by a hybrid protein, five additional short polypeptide fragments are formed along with the main product, making it difficult to further purify the target peptide.

The invention solves the problem of expanding the assortment of antimicrobial peptides and obtaining the antimicrobial peptide of arenicin.

The problem is solved by cultivating the producer strain, inducing the synthesis of recombinant protein, destroying cells, separating the insoluble fraction of the cell protein, affinity purification of the His8-TrxL-Ar2 fusion protein using metal chelate chromatography, solubilizing it, and splitting the residue with bromine in an acidic medium methionine, the separation of reaction products and the final purification of arenicin by reverse-phase HPLC.

To obtain a hybrid protein containing arsenicin, a new strain of the bacterium Escherichia coli BL21 (DE3) / pE-His8-TrxL-Ar2 is used. This producer strain is obtained by transforming competent cells of Escherichia coli strain BL-21 (DE3) with the plasmid pE-His8-TrxL-Ar2, consisting of a BglII / XhoI DNA fragment containing a T7 RNA polymerase transcription promoter, a ribosome binding site and a sequence a His8-TrxL-Ar2 hybrid polypeptide containing a histidine octamer, a modified thioredoxin A (M37L) and arenicin, and a BglII / XhoI DNA fragment of plasmid pET20b (+) containing a T7-RNA polymerase transcription terminator, a replication initiation site and a β- gene lactamase determining transf resistance rmirovannyh plasmid pE-His8-TrxL-Ar2 Escherichia coli cells to ampicillin resistance. The biosynthesis of the product is carried out as follows: cells of strain BL21 (DE3) / pE-His8-TrxL-Ar2 are grown in a rich medium (for example, LB) with the addition of 100-200 μg / ml ampicillin until the culture reaches an optical density of OD ₆₀₀ 0.5-1 , 0 at a temperature of 37 ° C, stepwise increasing the volume of the culture fluid by successive reseeding of the material, after which protein synthesis is induced with isopropyl-β-D-galactoside at a concentration of 0.1-1.0 mM or lactose and grow for another 5-6 hours at temperature 25-30 ° С. An increase in the yield of recombinant protein can be achieved by forced aeration of the culture fluid and the use of enriched culture media (for example, with the addition of glucose).

Obtaining His8-TrxL-Ar2 fusion protein from producer cells involves the following stages: separation of bacteria from the culture medium by centrifugation, their destruction by one of the commonly used methods (ultrasonic disintegration, chemical lysis using detergents and chaotropic agents), washing with inclusion bodies buffered solutions from water-soluble cell components, solubilization of inclusion bodies and purification of the hybrid protein using metal chelate chromatography under denaturing conditions.

Inclusion bodies obtained after biomass disintegration are processed according to the following scheme:

1. Inclusion bodies are washed twice with 50 mM phosphate buffer (pH 7.8) or 25 mM Tris-HCl buffer (pH 8.1).

2. The washed inclusion bodies are dissolved in 100 mM phosphate buffer (pH 7.8) or 50 mM Tris-HCl buffer (pH 8.1) containing 6 M guanidine-HCl and 10 mM imidazole. The precipitate was separated by centrifugation.

3. A solution containing the desired fusion protein is subjected to affinity chromatography on a resin with chelated Ni ²⁺ or Co ^{2+ ions} . As a washing solution, 100 mM phosphate buffer (pH 7.8) or 50 mM Tris buffer (pH 8.1) containing 6 M guanidine-HCl and 10 mM imidazole are used. As an eluting solution, a buffer containing 6 M guanidine-HCl and 0.5 M imidazole is used.

4. The eluate fraction containing the His8-TrxL-Ar2 fusion protein is dialyzed against deionized water at 4 ° C for 12-18 hours. The precipitate formed is collected and lyophilized.

5. A portion of purified His8-TrxL-Ar2 is dissolved in 80% trifluoroacetic acid, or in 70% formic acid, or in 6 M guanidine-HCl with the addition of 0.1 M HCl and an equal mass of bromine is added. The reaction mixture is incubated in the dark for 16-20 hours at a temperature of 20-25 ° C. The reaction is stopped by adding three to five times the volume of water, after which the sample is evaporated under vacuum at a temperature of 37 ° C. The procedure for adding water and evaporation is repeated at least three times.

6. A portion of the reaction products is dissolved in 10% acetonitrile with the addition of 0.1% trifluoroacetic acid and purified by reverse-phase HPLC in a concentration gradient of acetonitrile. The fraction containing arsenicin is freeze-dried.

7. If necessary, additional purification of the target product by HPLC can be carried out.

8. The identity of the recombinant arecinicin to the natural peptide is established by the methods of SDS-PAGE electrophoresis in the Tris-Tricin system, MALDI time-of-flight mass spectrometry, sequencing of the N-terminal amino acid sequence by the Edman method, as well as comparative testing of antimicrobial activity by serial dilution in a liquid nutrient medium. The degree of purification of arenicin is determined by the methods of PAGE-electrophoresis in Tris-tricin system and MALDI time-of-flight mass spectrometry.

The invention illustrates an example.

Example.

Obtaining arenicin.

The cultivation of cells of the producer strain of Escherichia coli BL21 (DE3) / pE-His8-TrxL-Ar2 is carried out as follows.

Cells of strain BL21 (DE3) / pE-His8-TrxL-Ar2 are grown in LB liquid medium with the addition of 150 μg / ml ampicillin and 1% glucose until the culture reaches an optical density of OD ₆₀₀ 1.0 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 5 hours at a temperature of 30 ° C.

After fermentation, the cells of the hybrid protein producer (biomass) are separated by centrifugation at 4000 g for 10 min, resuspended in lysis buffer (100 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 7.8), 0.5 M NaCl, 1 % Triton X-100) and destroyed using an ultrasonic homogenizer. All work on the isolation of the hybrid protein is carried out at a temperature of 4 ° C. The insoluble fraction of the cell protein (inclusion body) containing the fusion protein is separated by centrifugation. Inclusion bodies are resuspended twice in 50 mM phosphate buffer (pH 7.8) and centrifuged.

The washed inclusion bodies are dissolved at a concentration of 20 mg / ml in 100 mM phosphate buffer (pH 7.8) containing 6 M guanidine-HCl and 10 mM imidazole. The precipitate was separated by centrifugation. The supernatant is applied to a pre-balanced column with Ni-NTA agarose (Qiagen) at the rate of 1 g of total protein per 100 ml of resin. The column is washed with 100 mM phosphate buffer (pH 7.8) containing 6 M guanidine-HCl and 10 mM imidazole. The recombinant protein enriched fraction was eluted with 100 mM phosphate buffer (pH 7.8) containing 6 M guanidine-HCl and 0.5 mM imidazole and dialyzed against 100 volumes of deionized water at 4 ° C. for 18 hours through dialysis membrane retaining molecules with a mass of more than 1 kDa. The precipitate is collected and freeze-dried.

The analysis of fractions, as well as the degree of purity of the hybrid protein at different stages of isolation, is determined by means of PAGE electrophoresis in a Tris-Tricin buffer system and by MALDI time-of-flight mass spectrometry.

A portion of the purified fusion protein is dissolved in 80% trifluoroacetic acid at a concentration of 2%, 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 with constant shaking for 20 hours. The reaction is stopped adding five times the volume of deionized water, after which the sample is evaporated at 37 ° C. The procedure for adding water and evaporation is repeated three times.

A portion of the reaction products is dissolved in a concentration of 10 mg / ml in 10% acetonitrile with the addition of 0.1% trifluoroacetic acid. Under these conditions, both the desired peptide and the unsplit fusion protein and the carrier protein are completely dissolved. Insoluble precipitate was separated by centrifugation and discarded. Separation of the cleavage reaction products and purification of arenitsin is carried out using reverse phase HPLC on preparative columns in a system consisting of buffer A (5% acetonitrile, 0.1% TFA) and buffer B (80% acetonitrile, 0.1% TFA). Arenicin is eluted with a linear gradient of buffer B from 10 to 40%. Detection is carried out at a wavelength of 214 nm. The eluate fraction corresponding to arsenicin was collected and evaporated lyophilically. The yield of arenicin with a purity of not less than 99% obtained by the claimed method (without using a fermenter) is at least 5 mg / l of culture.

The amino acid sequence of the obtained peptide is identical to natural arsenicin (Arg ¹ -Trp ² -Cys ³ -Val ⁴ -Tyr ⁵ -Ala ⁶ -Tyr ⁷ -Val ⁸ -Arg ⁹ -Ile ¹⁰ -Arg ¹¹ -Gly ¹² -Val ¹³ -Leu ¹⁴ - Val ¹⁵ -Arg ¹⁶ -Tyr ¹⁷ -Arg ¹⁸ -Arg ¹⁹ -Cys ²⁰ -Trp ²¹ ). Automatic microsequencing on a Precise 491 cLC Protein Sequencing System (Applied Biosystems, USA) is used to determine the N-terminal amino acid sequence of arenicin. The identification of phenylthiohydantoin derivatives of amino acids is carried out on a 120A PTH Analyzer (Applied Biosystems, USA). 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 sequentially cleaving N-terminal amino acid residues in the form of phenylthiohydantoins and identifying cleaved amino acid derivatives by reverse-phase high-performance liquid chromatography. As a result of automatic microsequencing, the complete identity of the amino acid sequences of genetic engineering and natural arenicin was established.

MALDI (matrix-assisted lazer desorption ionization) mass spectrometric analysis using a VISION 2000 instrument (ThermoBioAnalysis, OK) is used to determine the molecular weight of arenicin. As a matrix using 0.15 M 2,5-dihydroxybenzoic acid in a mixture containing 25% methanol and 0.1% trifluoroacetic acid. The sample is irradiated with a UV laser with a wavelength of 337 nm and a maximum energy of 250 μJ in a pulsed mode with a frequency of 3 ns. In a mass spectrometric study of arenicin (laser power - 45%, the number of laser strokes - 69), a peak was obtained with m / z 2772 (see drawing), corresponding to the molecular ion of natural arsenicin (calculated molecular weight is 2772.35 Da).

The antimicrobial activity of arenicin is determined by the method of radial diffusion of peptides in an agarose gel with a test culture of the microorganism, which allows to evaluate the microbicidal activity, spending the minimum amount of the drug. To determine the antimicrobial activity of individual fractions of the polypeptides, cultures of the following microorganisms are used: Escherichia coli, strain ML-35p (gram-negative bacterium); Listeria monocytogenes, strain EGD (gram-positive bacterium); Candida albicans (Ascomycetes class mushroom). The antimicrobial effect of the selected peptides is fixed after 24 hours by the presence of zones of growth inhibition of the test culture (table 1). It was shown that genetically engineered and natural arenicins in the amount of ~ 1 μg exhibit microbicidal activity against all three microbes. The action of both peptides against gram-positive and gram-negative bacteria, as well as their antifungal activity, are almost identical. Thus, for example, the minimum inhibitory concentration of both natural and recombinant peptides with respect to E. coli is 25 μg / ml.

Table 1. Antimicrobial action of genetically engineered and natural arenicin Test microorganisms Arenicin genetic engineering natural Gram-positive bacterium Listeria monocytogenes, strain EGD + + Gram-negative bacterium Escherichia coli strain ML-35p + + Candida albicans ascomycete fungus + +

Claims (1)

A method of obtaining an antimicrobial peptide of arenicin, including culturing a recombinant producer strain of Escherichia coli BL21 (DE3) / pE-His8-TrxL-Ar2 in a nutrient medium, disrupting the obtained cells, isolating inclusion bodies containing His8-TrxL-Ar2 fusion protein, purification of the hybrid protein by affinity chromatography, its solubilization and cleavage with bromine cyan in an acidic medium according to the methionine residue, separation of the reaction products and purification of the target product by reverse-phase HPLC.