RU2619170C2 - RECOMBINANT PLASMID DNA pER-ARNS3, ENCODING FUSION PROTEIN CAPABLE OF AUTOCATALYTIC SPLITTING WITH FORMATION OF ARNS3, ESCHERICHIA COLI STRAIN C3030/pER-ARNS3 PRODUCER OF SAID PROTEINS AND METHODS FOR RECOMBINANT ARNS3 PRODUCTION - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention refers to biochemistry, namely to recombinant plasmid DNA pER-ARNS3 providing the synthesis of a hybrid polypeptide comprising ARNS3 and mini-intein Ssp DnaB, in Escherichia coli cells. The said plasmid DNA comprises BsaBI/Eco0109I-DNA plasmid fragment pTWIN-1 gene translation amplifier 10 of phage T7, NdeI/BamHI-DNA fragment containing the mini-intein gene Ssp DnaB and sequence of ARNS3 recombinant gene adapted to these sites. The plasmid also contains a b-lactamase gene that determines the resistance of E. coli cells transformed by plasmid pER-ARNS3 to penicillin antibiotics, as a genetic marker and unique restriction endonuclease recognition sites. This invention also discloses a strain of Escherichia coli C3030/pER-ARNS3, producing the said fusion polypeptide. This invention also discloses a method for production of the said ARNS3 recombinant protein, comprising culturing of the said strain of Escherichia coli C3030/pER-ARNS3.

EFFECT: invention provides recombinant ARNS3 with high yield according to a simplified technology.

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Description

The invention relates to biotechnology, in particular to genetic and protein engineering. It can be used to produce recombinant APHC3.

APCS3 is the analgesic actinia polypeptide of Heteractiscrispa. ARNS3 has trypsin-inhibiting activity, is able to inhibit the in vitro pain vanilloid receptor TRPV1, and also has an analgesic effect in vivo. The properties shown by him allow the use of this polypeptide for the treatment of diseases associated with inflammatory or neuropathological processes, as well as use as an analgesic agent.

APCS3 is a 56-membered polypeptide (SEQ ID NO 2).

A known method for the isolation of ARNS3 from a natural source (SA Kozlov, Ya, A. Andreev, A.N. Murashev, DI Skobtsov, IA D'yachenko, and EV Grishin. Russian Journal of Bioorganic Chemistry, 2009, vol. 35, No. 6 , pp. 711-719). The disadvantage of this method is the small product yields and the inability to scale the selection.

The prototype closest to the patented method for producing ARNS3 is described in (Yaroslav A. Andreev, Sergey A. Kozlov, Yuliya V. Korolkova, Igor A. Dyachenko, Dmitrii A. Bondarenko, Denis I. Skobtsov, Arkadii N. Murashev , Polina D. Kotova, Olga A. Rogachevskaja, Natalia V. Kabanova, Stanislav S. Kolesnikov, and Eugene V. Grishin. MarDrugs. 2013 Dec; 11 (12): 5100-5115). In this work, the peptide was obtained by a biotechnological method using the expression of a recombinant gene in bacteria. As a result of gene expression, a hybrid protein was synthesized consisting of the target peptide, thioredoxin and affinity tags. The disadvantage of this method is the need to use TEV proteinase to isolate APH3 from the fusion protein.

The invention solves the problem of obtaining highly productive recombinant bacterial producer strain, allowing to obtain recombinant ARHC3 in high yield and by simplified technology.

The problem is solved due to the fact that:

1. The expression plasmid pER-APHC3 DNA was constructed by cloning the APHC3 gene into the TWIN1 vector plasmid.

2. By transforming the plasmid DNA pER-APHC3 of Escherichia coli C3030 cells, a producer strain is obtained.

3. In the induced cultivation of the producer strain, the biosynthesis of the recombinant fusion protein DnaBAPHC3 takes place in the form of inclusion bodies containing, along with APCS3, the Ssp DnaB mini-intein sequence.

4. Cell biomass is destroyed in the buffer solution and a precipitate containing the APH3 fusion protein is isolated. The APPC3 fusion protein is extracted from inclusion bodies with a buffer solution (50 mM Tris / HCl, 2 M urea, 10 mM EDTA, 1 mM PMSF, 0.5 DTT, pH 10) centrifuged and the supernatant is isolated. The supernatant was dissolved in a renaturing buffer (100 mM Tris / HCl, 10 mM glycerol, 1 mM PMSF). The pH of the solution was adjusted with hydrochloric acid to 7.2, thereby initiating the autocatalytic cleavage of the hybrid protein, and incubated at 22 ° C for 48 hours. The pH of the solution was adjusted with hydrochloric acid to 3.0 and incubated at 8 ° C for 24 hours. The solution was centrifuged and the supernatant was applied to an ion exchange resin . The protein is eluted with a buffer step of 50 mM MES, 1.5 M NaCl, pH 5.5-5.6. Further purification and analysis of recombinant APCS3 is carried out by reverse phase chromatography. The identification of the resulting recombinant ARNS3 was carried out using mass spectrometry.

The technical result of the autocatalytic cleavage of the hybrid protein is the formation of APHC3, the yield of which reaches 10% relative to the total cell protein with a drug purity of at least 98%.

To avoid the difficulties associated with the cleavage of the recombinant fusion protein using various proteases, such as enterokinase, factor X, etc., as well as to reduce the cost of this stage, we used the mini-intein dnaB from Synechocystissp as part of the fusion protein. (Wu, N., Xu, M.-Q., Liu, X.-Q. (1998) Biochim. Biophys. Acta, 1387, 422-4327) for targeted autocatalytic cleavage of a hybrid into a target polypeptide and intein. For this purpose, the APHC3 gene (SEQ ID NO 1) is amplified by PCR using a plasmid with the artificial APHC3 gene as a template and cloned into the vector plasmid pTWIN1 containing the intein gene from Synechocystissp. dnaB (Wu, H., Xu, M.-Q., Liu, X.-Q. (1998) Biochim. Biophys. Acta, 1387, 422-432; Evans, J, TC, Bermer, J., Xu , M.-Q., (1999) J. Biol. Chem., 274, 18359-18363), at the restriction enzyme sites SapI and BamHI. The resulting expression plasmid pER-APHC3, the structure of which is shown in FIG. 1, transformed E. coli C3030 cells. The DnaBAPHC3 fusion protein resulting from expression of the recombinant gene is capable of autocatalytic cleavage into APHC3 and intein. The yield of high purity ARNS3 (98%) after reverse phase chromatography reaches 10% relative to the total cell protein.

The proposed technical solution uses a producer strain of Escherichia coli C3030 containing plasmid DNA pER-APHC3 for superproduction of the fusion protein DnaBAPHC3 containing the sequence APHC3 and intein Ssp dnaB.

Use recombinant plasmid DNA pER-APHC3

- the coding amino acid sequence of recombinant APHC3;

- consisting of:

The BsaBI / Eco0109I DNA fragment of the commercial plasmid pTWIN1 (NewEnglandBiolabs), containing the T7 RNA polymerase promoter and transcriptional terminator, T7 phage 10 gene translation enhancer, NdeI / BamHI DNA fragment containing the DnaB mini-intein gene and adapted to it recombinant APHC3 gene sequence,

- containing:

as a genetic marker, the β-lactamase gene, which determines the resistance of E. coli cells transformed with the plasmid pER-APHC3 to penicillin antibiotics;

unique recognition sites for restriction endonucleases located to the left of the BamHI-NdeI- site: 849 bp, XbaI - 888 bp, EcoRV - 2921 bp, HpaI - 2977 bp

The design of the recombinant plasmid DNA pER-APHC3 provides a high level of expression of the DnaBAPHC3 fusion protein gene cloned therein containing the APHC3 gene connected to the SspDnaB gene at the 3'-end. To construct a plasmid, a chemical approach is used, which allows the use of optimal regulatory elements that control its expression for the expression of the cloned structural gene.

The ARNS3 gene is obtained by amplification of a plasmid site (Yaroslav A. Andreev, Sergey A. Kozlov, Yuliya V. Korolkova, Igor A. Dyachenko, Dmitrii A. Bondarenko, Denis I. Skobtsov, Arkadii N. Murashev, Polina D. Kotova, Olga A. Rogachevskaja, Natalia V. Kabanova, Stanislav S. Kolesnikov, and Eugene V. Grishin. MarDrugs. 2013 Dec; 11 (12): 5100-5115).

Its terminal sections containing the restriction endonuclease sites corresponding to the vector were introduced by PCR with synthetic oligonucleotide primers A1 and Bl (SEQ ID NO 3) and then the gene was cloned into the vector plasmid pTWIN1.

The proposed producer strain Escherichia coli C3030 / pER-APHC3 is characterized by the following features.

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

Cultural signs. Cells grow well on simple nutrient media. When growing on Difco agar, the colonies are round, smooth, cloudy, shiny gray, the edge is even. When growing on liquid media (on a minimal medium with glucose or LB broth) they form an intense smooth turbidity.

Physico-biological signs. Cells grow at temperatures from 4 ° C to 40 ° C with optimum pH from 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 are resistant to penicillin antibiotics (up to 500 μg / ml).

The strain producing E. coli C3030 / pER-APHC3 differs from the recipient strain E.coli C3030 only by the presence of recombinant plasmid DNA pER-APHC3, which gives it resistance to penicillin antibiotics.

Producer strains are obtained by transformation of competent E. coli C3030 cells with the corresponding recombinant plasmid DNA.

E.coli C3030 / pER-APHC3 cells are superproducers. Upon induction with isopropylthio-β-D-galactoside, an effective biosynthesis of the DnaBAPHC3 fusion protein occurs, which accumulates in the cells in the amount of 20% of the total cell protein in soluble form.

The invention is as follows. Recombinant plasmid DNA pER-APH3 is constructed, for which the recombinant APH3 gene is amplified by PCR with synthetic oligonucleotide primers (SEQ ID NO 3) containing the restriction enzyme sites SapI (N-terminus of the gene, primer A1) and BamHI (C is the end of the gene, p B1), the DNA obtained is digested with the appropriate restriction enzymes and then ligated with the vector plasmid pTWIN1 digested at the same sites. The competent E. coli C3030 cells are transformed with a ligase mixture and plated on LB agar containing 50 μg / ml ampicillin or another penicillin antibiotic. The resulting clones are analyzed by sequencing.

The strain producing E. coli C3030 / pER-APHCS3 is grown in a rich medium (YT-, LB-broth, etc.) (or isopropylthio-β-D-galactoside is induced and grown again) until the culture density is reached.

(SEQIDNO 1) shows the structure of the recombinant APHC3 gene. The (SEQIDNO 2) depicts the amino acid sequence of recombinant APHC3.

The invention is illustrated by the following figures.

FIG. 1 - Physical map of the plasmid pER-APCS3.

FIG. 2 - Electrophormalizate of cells of the producer strain of E. coli C3030 / pER-APHC3.

FIG. 3 - Analytical HPLC profile of recombinant ARNS3.

FIG. 4 - Mass spectrometric analysis of recombinant ARNS3.

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA

Chemical synthesis of oligonucleotides is carried out by the solid-state phosphoamidite method on an ASM-102U DNA synthesizer (BIOSET, Novosibirsk) with the oligonucleotide chain being expanded from the 3'-end to the 5'-end using protected phosphamidites - 5'-dimethoxytrityl-N-acyl-2 '-deoxynucleoside-3'-O- (β-cyanethyl-diisopropylamino) -phosphites activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 μmol, using porous glass (pore size 500 Å) as a support, to which the first nucleoside unit (load 20-30 μmol / g) is connected via a 3'-succinate bond. The synthetic cycle of the standard phosphoamidite method is used.

To prepare the DNA vector of plasmid pTWTN1 (3 μg, 1 pmol) is treated in 40 μl of Y buffer (33 mm Tris-acetate, pH 7.9, 10 mm Mg-acetate, 66 mm K-acetate 1, 0.5 mm DTT, 0.1 mg / ml BSA) with the restriction enzyme SapI (10 units act.), And then in 40 μl of buffer R (10 mm Tris-HCl, pH 8.5, 10 mm MgCl ₂ , 100 mm KCl, 0.1 mg / ml BSA) with BamHII restriction enzyme (10 units act.) for 1 h at 37 ° C. The vector fragment after electrophoresis in a 15% agarose gel was excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 min and applied to a Nucleo Spin ExtractII column. Wash with NT 3 buffer and elute with 50 μl of NE buffer.

To prepare the APHC3 gene, amplification is carried out by PCR using a plasmid with the artificial APHC3 gene (0.01 μg in the sample) as a template, and synthetic oligonucleotides A1 and B1 (60 pmol each) as primers. PCR is carried out in a DNA amplifier, in a buffer solution consisting of each of four dNTPs at a concentration of 0.5 mM and 5 units. Act. Taq DNA polymerase, in the following mode: denaturation - 1 min at 94 ° С, annealing - 30 sec at 60 ° С, elongation - 40 sec at 72 ° С, 30 PCR cycles. The gene after electrophoresis in a 15% agarose gel is excised from the gel and transferred to 200 μl NT buffer, dissolved at 50 ° C for 5-10 minutes and applied to a Nucleo Spin ExtractII column. Wash with NT 3 buffer and elute with 50 μl of NE buffer, then digest with the same restriction enzymes used in the preparation of the vector and isolate the target fragment from the agarose gel.

The resulting synthetic fragment with the recombinant APHC3 gene in an amount of 2 pmol was added to a solution of 1 μg of the above vector fragment in 10 μl of buffer (20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiothreitol ) and are ligated with 10 units. Act. T4 DNA ligases for 12 hours at 10 ° C.

An aliquot of the reaction mixture is used to transform competent E. coli C3030 cells. Transformants are plated on plates with LB agar containing 50 μg / ml ampicillin. The DNA of plasmid pER-APCS3 was isolated from the clones and analyzed using endonucleases NdeI, BamHI. Screening of recombinants is carried out using sequencing. The physical map of the plasmid pER-APHC3 is shown in FIG. 1. Indicated restriction endonuclease sites. Ori is the site of initiation of replication of the tsamid. Ap ^R is an ampicillin resistance gene. Lad - lactose operon repressor gene. DnaBAPHC3 is a recombinant protein gene encoding APHC3 and DnaB intein.

Example 2

Obtaining a producer strain of E. coli C3030 / pER-APHC3 and determining its productivity

The strain producing E. coli C3030 / pER-APH3 is obtained by transformation of competent E. coli cells C3030 with the plasmid pER-APH3, as described in example 1.

The producer strain E. coli C3030 / pER-APHCS3 was grown at 37 ° C in 100 ml of LB broth (pH 7.0) with 50 μg / ml ampicillin for 2 hours on a shaker at a speed of 190 rpm to a turbidity of A ₅₅₀ 0.7-0.8, add isopropylthio-β-D-galactoside to a concentration of 0.2 mM and continue the process for another 6 hours, or continue to grow in the absence of an inductor for 6 hours. Each hour, a 2 ml sample is taken, A is determined ₅₅₀ and the amount of culture corresponding to 1 ml with A ₅₅₀ 1.0, centrifuged for 5 minutes at 6000 rpm. The precipitated cells in 100 μl of lysing buffer with dye bromphenol blue are treated with ultrasound for 20 seconds, heated for 3 min at 100 ° C and 4 μl samples are used for electrophoresis in 15% SDS-PAGE. The gel was stained with Coomassie R-250 according to a standard technique and scanned using a Shimadzu CS-930 densitometer. Figure 2. presents the electrogram of the cell lysate of the producer strain E.coli C3030 / pER-APH3, where M is the molecular weight standard, L is the cell lysate, C is the cleavage of the hybrid protein, 1 is a hybrid protein containing APHC3, 2 is Dnab intein .

Example 3

Obtaining a hybrid protein ARNC3, its autocatalytic cleavage and isolation of recombinant ARNC3

After fermentation, the cells of the hybrid protein producer (biomass) were separated by centrifugation (5000 g, 20 min, 4 ° C), destroyed on an ultrasonic disintegrator in a buffer (50 mM Tris / HCl, 10 mMEDTA, 1 mMPMSF) and isolated by centrifugation (15000 g, 45 min) precipitate. The fusion protein is extracted from inclusion bodies with a buffer solution (50 mM Tris / HCl, 2 M urea, 10 mM EDTA, 1 mM PMSF, 0.5 DTT, pH 10) centrifuged and the supernatant is isolated. The supernatant was dissolved in a renaturing buffer (100 mM Tris / HCl, 10 mM glycerol, 1 mM PMSF). The pH of the solution was adjusted with hydrochloric acid to 7.2, thereby initiating the autocatalytic cleavage of the hybrid protein, and incubated at 22 ° C for 48 hours. A sample of 30 μl was taken from the resulting mixture and heated with bromphenol blue dye for 3 minutes at 100 ° C. Samples of 4 μl are used for electrophoresis in 15% SDS-PAGE. The gel was stained with Coomassie R-250 according to a standard technique and scanned using a Shimadzu CS-930 densitometer. Ha FIG. 2 shows the cleavage of a hybrid protein. After incubation at 22 ° C for 48 h, the pH of the solution was adjusted with hydrochloric acid to 3.0 and incubated at 8 ° C for 24 h. It was centrifuged and the supernatant was applied to an ion-exchange resin. The protein is eluted with a buffer step of 50 mM MES, 1.5 M NaCl, pH 5.5-5.6. Further purification and analysis of recombinant APCS3 is carried out by reverse phase chromatography. The resulting recombinant APHC3 is identified using TOF-ESI mass spectrometry using an Agilenttechnologies 6224 mass spectrometer. The resulting recombinant APHC3 signal corresponds to a calculated mass value of 6111 Da. In FIG. 3 shows the profile of analytical RP HPLC ARNS3. In FIG. 4 presents the mass spectrum of ARNS3.

Example 4

Testing the APC3 analgesic activity in mice in the hot plate test

Mice are divided into 2 groups (control and experimental), 10 animals each. Tests are carried out on male CD-1 mice weighing 20-30 g. Painful stimulation is modeled by placing the mouse on a metal surface heated to 55 ° C. To prevent paw burns, the exposure time is limited to 180 seconds. Test samples are dissolved in sterile saline and administered at a dose of 0.1 mg / kg intravenously. The analgesic effect is measured by the increase in the elapsed time from the moment the animal was placed on the plate until the first licking of the hind paw. Measurement of pain sensitivity is carried out 15 minutes after administration of the drug. The results are processed statistically, the significance of differences in the results of the control and experimental groups is determined using ANOVA and Tukey test. The resulting recombinant peptide at a given concentration increases the measured time by at least 50%.

Claims (3)

1. Recombinant plasmid DNA pER-APHC3 for expression of a hybrid DnaBAPHC3 polypeptide containing APHC3 having the amino acid sequence of SEQ ID NO: 2 and mini-intein Ssp DnaB containing the BsaBI / Eco0109I plasmid pTWIN-1 DNA fragment containing the promoter and terminator of the transcript T7 RNA polymerase, translation enhancer of the T7 phage 10 gene, NdeI / BamHI DNA fragment containing the Ssp DnaB mini-intein gene and a recombinant APCS3 gene sequence adapted to these sites having the sequence SEQ ID NO: 1, β-lactamase gene, determining stability of tra penicillin antibiotics formed by the plasmid pER-ARNS3 of E. coli as a genetic marker and unique restriction endonuclease recognition sites located at the next distance to the left of the BamHI-NdeI site - 849 bp, Xba - 888 bp, EcoRV - 2921 bp; HpaI - 2977 bp 2. The strain Escherichia coli C3030 / pER-APHC3 producing a hybrid DnaBAPHC3 polypeptide containing the amino acid sequences of APHC3 and mini-intein Ssp DnaB obtained by transforming the cells of the Escherichia coli C3030 strain of the recombinant plasmid DNA pER - APHC3 according to claim 1. 3. A method for producing recombinant APHC3 with SEQ ID NO: 2, comprising transforming the Escherichia coli C3030 strain of the obtained pER-APHC3 plasmid DNA according to claim 1, culturing the resulting Escherichia coli C3030 / pER-APHC3 producing strain of claim 2, separating the hybrid protein in the form of inclusion bodies after cell disruption, extraction of the hybrid protein from inclusion bodies, dilution in renature buffer, incubation of the solution at a pH of 7.2 and a temperature of 22 ° C, incubation of a protein solution at 8 ° C and a pH of 3.0, centrifugation, supernatant separation and chromatograph ical desired product purification.

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