RU2316590C1 - RECOMBINANT PLASMID DNA pE-His8-TrxL-Ar2 ENCODING FUSION PROTEIN COMPRISING ANTIMICROBIAL SEA ANNELID WORM Arenicola marina PEPTIDE ARENICIN AND STRAIN Escherichia coli BL21(DE3)/pE-His8-TrxL-Ar2 AS PRODUCER OF ARENICIN-CONTAINING FUSION PROTEIN - Google Patents

Abstract

FIELD: biotechnology, genetic and protein engineering.

SUBSTANCE: method involves construction of recombinant plasmid DNA pE-His8-TrxL-Ar2 of size 4059 base pairs encoding fusion protein His8-TrxL-Ar2 comprising antimicrobial peptide arenicin consisting of Bgl II/Xho I DNA fragment comprising transcription promoter T7-RNA-polymerase, ribosome binding site and sequence of fusion polypeptide His8-TrxL-Ar2 comprising histidine octamer, modified thioredoxine A(M37L) and arenicin, and Bgl II/Xho I DNA-fragment of plasmid pET20b(+) comprising transcription terminator T7-RNA-polymerase, replication initiation site and β-lactamase gene determining resistance of Escherichia coli cells transformed with plasmid pE-His8-TrxL-Ar2 to ampicillin as a genetic marker. Also, method involves preparing the strain Escherichia coli BL21(DE3)/pE-His8-TrxL-Ar2 as a producer of fusion protein comprising antimicrobial peptide arenicin. Invention can be used in medicine and pharmaceutical industry.

EFFECT: valuable biological properties of DNA and strain.

3 cl, 2 dwg, 2 ex

Description

The invention relates to the field of biotechnology, genetic and protein engineering and can be used in medicine and in the pharmaceutical industry.

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 for stopping general and local infectious processes of various etiologies, disinfecting surgical instruments, probes, prostheses.

Due to the relatively low content of arenicin in a natural source, it is advisable to conduct large-scale structural and functional studies and clinical trials of this substance using artificial analogues obtained by chemical synthesis or recombinant expression. Due to the sufficiently large length of the peptide chain, the chemical synthesis of arenicin is expensive and cannot be considered as the basis for scaling up production. The difficulties of the genetic engineering approach, in turn, are due to the cytotoxicity of arenicin, which requires mandatory neutralization to achieve a satisfactory level of expression. In most of the previously created recombinant expression systems of other antimicrobial peptides, this problem was solved, first of all, by constructing less toxic hybrid proteins. However, the task of creating such a system for recombinant expression of arenicin has not been previously solved.

A known system for the expression of the antimicrobial peptide Cecropin A, including strain E. coli MC1061, transformed with the plasmid pCAMG, in which a fragment of L-ribulokinase is used as a partner for expression. The disadvantage of the system is the non-identity of the C-terminal amino acid residue of the resulting peptide to the corresponding residue of natural cecropin A, as a result of which the spectrum of its antimicrobial activity is narrowed [Callaway JE, Lai J., Haselbeck B., Baltaian M., Bonnesen SP, Weickmann J., Wilcox G ., Lei SP // Modification of the С terminus of cecropin is essential for broad-spectrum antimicrobial activity. Antimicrobial Agents and Chemotherapy, 1993, v. 37 (8), p.1614-1619].

Known closest to the claimed system for the expression of the antimicrobial adenoregulin peptide, which is a strain of E. coli BL21 (DE3), transformed with plasmid pET32a-adr, obtained on the basis of plasmid pET32a [Zhou YX, Cao W., Luo QP, Ma YS, Wang JZ Wei DZ // 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 presence of an additional methionine residue in the natural sequence of thioredoxin A and four methionine residues in the linker connecting the thioredoxin A to 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 constructing a plasmid that determines the synthesis of the recombinant peptide in the composition of the hybrid protein, and create a highly productive producer strain that allows to obtain His8-TrxL-Ar2 hybrid protein in high yield.

The problem is solved by constructing a recombinant plasmid DNA pE-His8-TrxL-Ar2 with a size of 4059 bp encoding a fusion protein His8-TrxL-Ar2 containing the antimicrobial peptide arsenicin, consisting of a BglII / XhoI-DNA fragment (figure 1) containing a T7 RNA polymerase transcription promoter, a ribosome binding site and a sequence of 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 T7 RNA polymerase transcription terminator, CA t initiation of replication and the β-lactamase gene, which determines the resistance of ampicillin to Escicichia coli transformed with plasmid pE-His8-TrxL-Ar2, as a genetic marker, and also by creating Escherichia coli strain BL21 (DE3) / pE-His8-TrxL- Ar2 is a producer of a hybrid protein containing the antimicrobial peptide arenicin.

An advantage of the present invention consists, firstly, in the use of a set of codons that are optimal for protein production in Escherichia coli in the chemical-enzymatic synthesis of the arenicin gene. Figure 1 shows the amino acid sequence of the His8-TrxL-Ar2 fusion protein containing arsenicin and the corresponding nucleotide sequence of the gene. The introduction of the methionine residue immediately before the N-terminal residue of the recombinant arenicin allows selective chemical cleavage of the polypeptide chain with bromine cyan to release the target peptide that does not contain any additional amino acid residues compared to natural arenicin. The use of thioredoxin A as a partner for recombinant expression can neutralize the toxicity of the antimicrobial peptide for the producer strain and thereby achieve high yields of the hybrid protein. The advantage of replacing leucine with the methionine residue at position 37 of natural thioredoxin A, as well as using the plasmid pET20b (+) as the basis for the creation of pE-His8-TrxL-Ar2, is to reduce the number of individual fragments resulting from the cleavage of the hybrid protein with bromine cyan in an acidic medium, which, in turn, facilitates further purification of the target peptide. The cleavage of the His8-TrxL-Ar2 fusion protein with cyanogen bromide gives only one by-product, which differs significantly in molecular weight and physicochemical properties from the target peptide, which facilitates the solution of the problem of its isolation. The presence of a histidine octamer at the N-terminus of the expressed sequence allows affinity purification of the His8-TrxL-Ar2 fusion protein by metal chelate chromatography. For the biosynthesis of a recombinant protein, optimal regulatory elements that control its expression are used: the T7-1ac promoter to prevent basal gene expression until the beginning of induction and a high level of transcription of the corresponding mRNA during induction, a highly efficient terminator of T7 phage transcription, and a block of stop codons excluding elongated biosynthesis variants of recombinant arenicin. The advantages of the proposed E. coli BL21 (DE3) strain are the use of bacteria with the Lon OmpT phenotype, which eliminates the possibility of proteolytic cleavage of the synthesized de novo recombinant 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 use of the T7-lac promoter and T7 terminator in the pE-His8-TrxL-Ar2 plasmid, provides fast and efficient protein production by E. coli cells upon isopropylthio-β- induction D-galactoside or lactose.

The construction of a new gene encoding the His8-TrxL-Ar2 fusion protein is carried out on the basis of plasmid pET20b (+). An artificial gene encoding a histidine octamer, modified thioredoxin A (M37L), and arenicin flanked by restriction enzyme sites BglII and XhoI are obtained by chemical synthesis of a set of oligonucleotide fragments, followed by their assembly and amplification by polymerase chain reaction (PCR). Before doping to obtain sticky ends, the amplification and vector plasmid is treated with restriction enzymes BglII and XhoI. The ligase mixture is used to transform competent E. coli DH10B cells. The selection of positive clones is carried out using PCR using specific primers and subsequent restriction analysis of the isolated plasmid DNA. The structure of the gene encoding a hybrid protein containing arsenicin is determined by Sanger sequencing.

Recombinant plasmid DNA pE-His8-TrxL-Ar2 encoding the His8-TrxL-Ar2 fusion protein containing the antimicrobial peptide arsenicin is characterized by the following features:

- has a molecular size of 4059 bp .;

- encodes a His8-TrxL-Ar2 fusion protein containing a histidine octamer, modified thioredoxin A (M37L) E. coli, and arenicin;

- consists of: a BglII / XhoI DNA fragment containing a T7 RNA polymerase transcription promoter, a ribosome binding site and a sequence of His8-TrxL-Ar2 hybrid polypeptide containing a histidine octamer, modified thioredoxin A (M37L) and arenicin, and BglII / XhoI a DNA fragment of plasmid pET20b (+) containing the T7 RNA polymerase transcription terminator, a replication initiation site and a β-lactamase gene;

- has a unique combination of features: promoter and terminator of transcription of RNA polymerase of bacteriophage T7; ribosome binding site; an artificial gene encoding a His8-TrxL-Ar2 fusion protein containing a histidine octamer, a modified thioredoxin A (M37L), and arenicin; β-lactamase gene that determines the stability of plasmid-transformed pE-His8-TrxL-Ar2.

To obtain a producer strain of the His8-TrxL-Ar2 fusion protein, pE-His8-TrxL-Ar2 plasmid DNA is used to transform competent Escherichia coli BL21 (DE3) cells and select clones possessing the ability to express the recombinant protein. The level of expression of recombinant proteins is controlled by denaturing PAGE electrophoresis.

The resulting producer strain Escherichia coli BL21 (DE3) / pE-His8-TrxL-Ar2 is characterized by the following features.

Morphological signs: rod-shaped cells, gram-negative, non-spore.

Cultural traits: when growing on LB agar medium, the colonies are round, smooth, cloudy, shiny, gray, the edge is even. When growing on liquid media (on a minimal medium with glucose or YT-broth) they form an intense smooth haze.

Physico-biological characteristics: cells grow at a temperature of from 4 ° C to 40 ° C with an optimum pH of 6.8 to 7.5. As a source of nitrogen, both mineral salts in the ammonium form and organic compounds in the form of peptone, tryptone, yeast extract, amino acids, etc. are used. Amino acids, glycerin, carbohydrates are used as a carbon source.

Antibiotic resistance: cells show resistance to ampicillin (up to 500 μg / ml) due to the presence of the β-lactamase (bla) gene in the plasmid.

The advantages of the proposed producer strain are the use of bacteria with the Lon OmpT phenotype, which excludes the possibility of proteolytic cleavage of the synthesized de novo recombinant 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 use of the T7-lac promoter and T7 terminator in the pE-His8-TrxL-Ar2 plasmid, provides fast and efficient protein production by E. coli cells upon isopropylthio-β- induction D-galactoside or lactose.

E. coli BL21 (DE3) / pE-His8-TrxL-Ar2 cells are superproducers. Upon induction with isopropylthio-β-D-galactoside, an efficient biosynthesis of the His8-TrxL-Ar2 fusion protein, containing arsenicin, which accumulates in the cells in an amount of 20-25% of the total bacterial protein, occurs.

Figure 1 shows the nucleotide sequence of a BglII / XhoI DNA fragment containing a T7 RNA polymerase transcription promoter, a ribosome binding site, and a His8-TrxL-Ar2 hybrid polypeptide sequence; the corresponding amino acid sequence of His8-TrxL-Ar2; shows the natural nucleotide and amino acid sequences of thioredoxin A of E. coli and arenicin-2 A.marina. Figure 2 presents the physical map of the plasmid pE-His8-TrxL-Ar2.

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA pE-His8-TrxL-Ar2.

The nucleotide sequence corresponding to the His8-TrxL-Ar2 gene is obtained by enzymatic-chemical synthesis. The chemical synthesis of oligonucleotides used to assemble the nucleotide sequence is performed by the solid-phase phosphoamidite method with the oligonucleotide chain being extended from the 3′-end to the 5′-end using protected phosphamidites - 5′-dimethoxytrityl-N-acyl-2′-deoxynucleoside-3 '-O- (β-cyanoethyl diisopropylamino) -phosphites activated by tetrazole. The fragment encoding the modified thioredoxin A (M37L) is obtained by PCR amplification and directed mutagenesis using gene-specific primers using the E. coli thioredoxin A gene as the starting matrix. The remaining sections of the sequence are obtained by sequential annealing and elongation of mutually overlapping oligonucleotides. 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 DNA strip of about 570 bp. PEG-6000 solution was isolated from the gel by electroelution in a 15% solution and used in the ligase reaction with a DNA fragment of about 3.5 thousand bp obtained by treating plasmid pET20b (+) with BglII and XhoI restriction enzymes. As a result of the ligase reaction, a ring covalently closed DNA of 4059 bp is formed. The ligase mixture is used to transform competent E. coli DH-10B cells prepared according to the standard protocol using 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." Selected clones are used for growing in a liquid medium and isolation of plasmid DNA, which is analyzed for the presence of the 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 doped insert is completely identical to the originally planned one (Fig. 1).

Example 2

Obtaining a producer strain of E. coli BL21 (DE3) / pE-His8-TrxL-Ar2 producing the recombinant protein His8-TrxL-Ar2, and determining its productivity.

E. coli BL21 (DE3) cells are transformed with the plasmid pE-His8-TrxL-Ar2, as described in Example 1. Several colonies are transferred in a 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 thermostatically controlled shaker with a rotation speed of 200 min ^-1 at a temperature of 37 ° C, an aliquot of the culture is selected for subsequent analysis, an inducer of isopropylthio-β-D-galactoside is added to a concentration of 0.2 mM and incubation is continued at a temperature of 27 ° C in for 5 hours, taking 0.3 ml samples every hour to determine the optical density ti OD ₆₀₀ and subsequent electrophoretic analysis. Equal aliquots of the cell suspension, taken before introducing the inducer and after completion of growth, are centrifuged, the supernatant is separated and the cell pellet is analyzed by PAGE by electrophoresis under denaturing conditions. For this, samples are lysed with a buffer containing 1% SDS, 1% β-mercaptoethanol, 8M urea in a water bath for 5 min. Electrophoresis is carried out in 15% SDS-PAGE. The gel is stained with 0.025% Coomassie G-250 according to standard procedures. The appearance of a distinct band at 16 kDa in the samples taken after induction indicates the ability of the strain to synthesize recombinant His8-TrxL-Ar2. The relative content of the recombinant protein is determined by scanning and densitometric analysis of stained gels.

Claims (2)

1. Recombinant plasmid DNA pE-His8-TrxL-Ar2 with a size of 4059 bp encoding a fusion protein His8-TrxL-Ar2 containing the antimicrobial peptide arsenicin, consisting of The BglII / XhoI DNA fragment of FIG. 1, containing a T7 RNA polymerase transcription promoter, a ribosome binding site, and a His8-TrxL-Ar2 hybrid polypeptide sequence containing a histidine octamer, a modified thioredoxin A (M37L), and arenicin, The BglII / XhoI DNA fragment of plasmid pET20b (+), containing the T7 RNA polymerase transcription terminator, the replication initiation site and β-lactamase gene, which determines the resistance of ampicillin to Escicichia coli cells transformed with the pE-His8-TrxL-Ar2 plasmid, as a genetic marker. 2. The strain Escherichia coli BL21 (DE3) / pE-His8-TrxL-Ar2 is a producer of a hybrid protein containing the antimicrobial peptide arenicin.

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