RU2453602C2 - RECOMBINANT PLASMID DNA pET22b(+)/SLURP-1 CODING SLURP-1 PROTEIN, AND STRAIN OF BACTERIA Escherichia coli BL21(DE3)/pET22b(+)/SLURP-L-PRODUCENT OF HUMAN SLURP-1 PROTEIN - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: SLURP-1 peptide is prepared by transformation of competent cells Escherichia coli BL21 (BE3) by recombinant plasmid DNA pET22b(+)/SLURP-1.

EFFECT: invention provides higher effectiveness of SLURP-1 protein synthesis in cells Escherichia coli.

2 cl, 3 dwg, 1 tbl, 6 ex

Description

The invention relates to biotechnology, in particular to genetic engineering, in particular, to the production of the human protein SLURP-1, which can be used in medicine as a medicine.

Known regulatory proteins of the SLURP family, endogenous modulators of the nicotinic acetylcholine receptor (nAHR) activity found in human blood plasma and urine [Adermann K, Wattler F, Wattler S, Heine G, Meyer M, Forssmann WG, Nehls M. (1999) Protein Sci . 8 (4): 810-9], as well as in the cells of the skin and mucous tissue [Mastrangeli R, Donini S, Kelton CA, He C, Bressan A, Milazzo F, Ciolli V, Borrelli F, Martelli F, Biffoni M, Serlupi -Crescenzi O, Serani S, Micangeli E, El Tayar N, Vaccaro R, Renda T, Lisciani R, Rossi M, Papoian R. (2003) Eur J Dermatol. 13 (6): 560-70]. These proteins possess all the characteristic structural features of snake venom neurotoxins: they contain a leucine residue at the N-terminus of the molecule and 10 cysteine residues forming a toxin-like system of 5 disulfide bonds. Proteins of the SLURP family have functional and biological properties that are very interesting from a pharmacological point of view. Unlike alpha-neurotoxins, these proteins do not block in the presence of agonists (acetylcholine or nicotine), but rather activate the nAChR channel [Chimienti F, Hogg RC, Plantard L, Lehmann C, Brakch N, Fischer J, Huber M, Bertrand D , Hohl D. (2003) Hum Mol Genet 12 (22): 3017-24].

A known method for producing human SLURP-1, based on the expression in transformed mammalian cells of HEK 293T [Chimienti F, Hogg RC, Plantard L, Lehmann C, Brakch N, Fischer J, Huber M, Bertrand D, Hohl D. (2003) Hum Mol Genet. 12 (22): 3017-24]. The disadvantage of this method is the extremely low yield of the target product (0.1 mg protein with 1 liter of bacterial culture).

The invention solves the problem of producing recombinant human protein SLURP-1.

The problem is solved by constructing a recombinant plasmid DNA pET22b (+) / SLURP-1 encoding a human protein SLURP-1 (5660 bp), and the bacterial strain Escherichia coli BL21 (DE3) / pET22b (+) / SLURP-1 - producer of human SLURP-1 protein, which ensures the synthesis of this protein with an expression level of at least 10% of the total cellular protein.

Recombinant plasmid DNA pET22b (+) / SLURP-1 encoding a human SLURP-1 protein is characterized by the following features (Figure 1):

has a molecular weight of 5660 bp;

encodes the amino acid sequence of human SLURP-1;

contains: lac-promoter of E. coli, bla β-lactamase gene, which determines the resistance of ampicillin transformed by the plasmid pET22b (+) / SLURP-1, the ori site of replication initiation; unique recognition sites by restriction endonucleases having the following coordinates: XhoI - 158, HindIII - 173, EcoRI - 192, BamHI - 198, NdeI - 430, BglII - 534, MluI - 1256, PstI - 4495.

The expression of the human SLURP-1 gene in E. coli cells is accompanied by the accumulation of an artificial neuromodulator in the bacterial cytoplasm in the form of insoluble inclusion bodies. To isolate it, the use of denaturing agents is necessary.

To obtain a producer strain of a polypeptide with a human SLURP-1 structure, competent Escherichia coli BL21 (DE3) cells are transformed with the recombinant plasmid pET22b (+) / SLURP-1.

The resulting strain of Escherichia coli BL21 (DE3) / pET22b (+) / SLURP-1 is characterized by the following features.

Morphological signs. The cells are small, rod-shaped, gram-negative, non-spore-bearing, 1 x 3-5 μm, motile.

Cultural signs. When growing on a dense LA medium, the colonies are round, smooth, translucent, shiny, gray, the edge is even, the diameter of the colonies is 1-3 mm; pasty consistency. Growth in a liquid LB medium is characterized by even turbidity with the formation of a light precipitate.

Physico-biochemical characteristics. Cells grow at a temperature of 4-42 ° C with an optimum pH of 6.8-7.2. 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 are used. Amino acids, glycerin, carbohydrates are used as a carbon source.

Antibiotic resistance. Cells are resistant to ampicillin (up to 200 μg / ml) due to the presence of the beta-lactamase gene in the plasmid.

Strain E. coli BL21 (DE3) / pET22b (+) / SLURP-1 provides inducible synthesis of human SLURP-1 protein in an amount of at least 10% of the total cellular protein.

The resulting recombinant protein SLURP-1 can be used to treat psoriasis, cancer, and some forms of epilepsy.

The invention is illustrated by graphic materials:

Figure 1 presents the physical map of the recombinant plasmid pET22b (+) / SLURP-1;

figure 2 - electrophoregram of cell lysates of the strain of the recipient of E. coli BL21 (DE3) (lane 2), the producer strain of E. coli BL21 (DE3) / pET22b (+) / SLURP-1 (lane 3) in 13% - Mr. polyacrylamide gel, lane 1 - protein markers of molecular weight; the arrow indicates the protein SLURP-1;

figure 3 - purification of recombinant SLURP-1 after renaturation by HPLC on a JupiterC4 column (4.6 × 150, mm, Phenomenex, USA) in a linear gradient of acetonitrile concentration from 10% to 45% in 30 min in the presence of 0.1% TFA, the elution rate is 0.3 ml / min. Registration of the optical density of the eluate at 210 nm;

The invention is illustrated by the following examples.

Example 1. Construction of the gene SLURP-1 person.

The gene for the SLURP-1 protein containing an additional methionine residue at the N-terminus is constructed from 6 synthetic overlapping oligonucleotides using three-stage PCR. In the first stage, as a result of successive amplification of primers 1 (SEQ No. 1) and 2 (SEQ No. 2), 3 (SEQ No. 3) and 4 (SEQ No. 4), 5 (SEQ No. 5) and 6 (SEQ No. 6) produce PCR fragments A, B and C, respectively. In the second stage, fragments A and B are amplified using primers 1 and 4 to obtain a PCR product containing fragment D. Fragments B and C are amplified using primers 3 and 6 to obtain a PCR product containing fragment D. At the final stage, fragments G and D amplified using primers 1 and 6 to obtain a fragment containing the SLURP-1 gene. In all PCR, the reaction mixture in a volume of 50 μl contains 140 ng of primers, 10 nmol of each of the four deoxynucleoside triphosphates and 1 unit. Act. Pfu DNA polymerase in Pfu polymerase buffer: 20 mM Tris-HCl, 2 mM MgSO ₄ , 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton X-100, 200 μg / ml bovine serum albumin (BSA), pH 8.75. The amplification cycle includes DNA denaturation at 95 ° C (1 min), annealing at 52 ° C (1 min) and elongation at 68 ° C (1 min). A total of 30 reaction cycles are carried out. The reaction mixture was applied to an agarose gel for electrophoresis separation and the desired fragments were excised from the gel with a scalpel, then the DNA was extracted from the gel using the Wizard SV Gel Clean Up System (Promega).

Example 2. Construction of recombinant plasmid DNA pET22b (+) / SLURP-1.

50 ng of a PCR DNA fragment containing the SLURP-1 gene (SEQ No. 7) is sequentially treated with restriction enzymes NdeI and BamHI (Fermentas, Lithuania), and a fragment of 257 bp is isolated from the obtained hydrolyzate in a 2% agarose gel, containing the SLURP-1 gene.

2 μg of plasmid DNA pET22b (+) was sequentially treated with restriction enzymes NdeI and BamHI, and vector DNA 5403 bp was isolated from the obtained hydrolyzate in 1% gel of low-melting agarose.

The resulting fragment and vector DNA are combined using a ligase reaction in 10 μl of ligation buffer (Fermentas) containing 1 unit T4 DNA ligase. 1 μl of the reaction mixture was used to transform 100 μl of competent XL-1 Blue cells. 1/10 of the cells used for transformation are seeded on LB agar containing 100 μg / ml ampicillin. From the grown clones, the target plasmid DNA pET22b (+) / SLURP-1 is isolated and analyzed by treatment with a set of restriction endonucleases NdeI and BamHI, followed by electrophoretic analysis of the lengths of restriction fragments in a 2% agarose gel.

Finally, the structure of the recombinant pET22b (+) / SLURP-1 DNA is confirmed by determining the nucleotide sequence in the region of an inserted fragment containing the synthetic human SLURP-1 gene.

Example 3. Obtaining and determining the productivity of the producer strain SLURP-1 person.

Recombinant plasmid DNA pET22b (+) / SLURP-1 transform competent cells of Escherichia coli BL21 (DE3) (Novagen). For this, 20 ng of pET22b (+) / SLURP-1 plasmid DNA is added to 200 μl of competent cells, mixed well and incubated in ice for 30 minutes. Then the cells are subjected to heat shock for 90 seconds at 42 ° C, after which they are again placed in ice and incubated for 4-5 minutes. Then 300 μl of SOB medium is added to the cell suspension and the cells are incubated for 60 min at 37 ° C and good aeration. Then, cells were plated in 100 μl per Petri dish with nutrient solid medium containing ampicillin (100 μg / ml). The result is a producer strain of human SLURP-1.

Colonies with E. coli BL21 (DE3) / pET22b (+) / SLURP-1 cells collected from Petri dishes were resuspended in 50 ml of Terrific Brouth (TB) liquid medium containing ampicillin (100 μg / ml). The cultivation is carried out at 37 ° C with moderate stirring (250 rpm./min.) To achieve a cell density in the culture corresponding to the absorbance value at 600 nm of 0.6. The cell culture is then transferred to 1 L of TB and continued to grow in a fermenter (Bioflow 3000, New Bronswick Scientific, USA). Cultivation is carried out at 37 ° C under conditions of automatic maintenance of the relative oxygen content in the system of at least 30% of the maximum achievable. Adjustable parameters are the speed of the mixer and the air flow rate. SLURP-1 gene expression is induced by the addition of isopropylthio-β-D-galactopyranoside to a final concentration of 0.025 mM at a cell density in culture corresponding to an absorbance value of 600 nm 1.0. After induction, cell cultivation is carried out for 8 hours.

A 1 ml sample was taken and centrifuged for 5 min at a speed of 6000 rpm, after which the cells were suspended in 100 μl of buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol, 0.005 % bromphenol blue, incubated for 10 min in a boiling water bath, samples with a volume of 5 μl were analyzed by electrophoresis in 13% polyacrylamide gel with sodium dodecyl sulfate. The gel is stained with Coomassie G-250 (FIG. 2), scanned and the percentage of recombinant protein in lysates is calculated using the Scion Image program (Scion Corp., USA). According to the scan protein SLURP-1 is 10% of the total cellular protein (figure 2).

Example 4. Isolation of recombinant human SLURP-1 protein.

Cells are harvested by centrifugation (10,000 g, 20 min, 4 ° C) and resuspended in cold 20 mM Tris / HCl buffer, 100 mM NaCI, 1 mM EDTA, pH 8.0 in a ratio of 10 ml of buffer per g of pellet. Next, the cell suspension is disintegrated by ultrasound (Branson Digital Sonifier, USA) at an output power of 50 W and 4 ° C for 5 minutes. The resulting suspension is centrifuged at 36,000 g for 20 minutes. The precipitate formed is resuspended in the initial buffer with the addition of 2 M urea. The suspension is transferred to a chilled glass, disintegrated for 10 seconds and centrifuged under the same conditions as before. The procedure is repeated twice. After washing with 2 M urea, the precipitate was washed twice with the initial buffer containing 1% Triton X-100, and then twice with deionized water (MilliQ, Millipore, USA). The washed inclusion bodies are stored at -70 ° C.

The washed inclusion bodies are resuspended in 30 mM Tris-glycine buffer, pH 9.2, containing 8 M urea (Carl Roth, Germany), at the rate of 10 ml of buffer per 1 g of inclusion bodies. The suspension is disintegrated at an output power of 50 W and 4 ° C for 30 seconds. Then, the suspension of inclusion bodies is sulfonated by adding 0.4 M sodium sulfite, cystine to a concentration of 0.4 mM and NiCl ₂ to a concentration of 5 mM. In this form, the bodies are left for 18 hours in an open glass with stirring and 25 ° C. The mixture is then centrifuged at 36,000 g for 1 hour and the supernatant is diluted 10 times with 2 M urea. After that, sulfonated SLURP-1 is applied to a DEAP-Spheronite-OH column, previously equilibrated with 30 mM Tris-HCl, pH 8.0 (buffer A). SLURP-1 was eluted with buffer A containing 300 mM NaCI and 8 M urea.

Dithiothreitol was added to the protein fraction containing SLURP-1 to a final concentration of 50 mM and further purified by reverse phase HPLC using a Jupiter C4 chromatographic column, А300, 10 × 250 mm (Phenomenex, USA) on a Smartline instrument (Knauer, Germany ) Elution of SLURP-1 is carried out with a gradient of acetonitrile from 10% to 45% for 30 minutes in the presence of 0.1% trifluoroacetic acid. The resulting preparation SLURP-1 with reduced disulfide bonds is lyophilized.

Example 5. Renaturation of recombinant human SLURP-1 protein.

Lyophilized and reconstituted SLURP-1 was dissolved in a renaturation buffer containing 50 mM Tris / HCl, 2 M urea, 0.5 M L-arginine, 4 mM GSH and 1 mM GSSG, pH 7.0, to a final protein concentration of 0.1 mg / ml. Renaturation is carried out at 4 ° C for 6 days.

The SLURP-1 solution was concentrated 4 times on a concentration cell with a membrane pore size of 1 kDa (Millipore, USA). The renatured SLURP-1 was purified by reverse phase HPLC on a Jupiter C4, A300, 4.6 x 250 mm column (Phenomenex, United States). Elution vdLinks-1 spend a gradient of acetonitrile from 20% to 45% for 30 min in the presence of 0.1% trifluoroacetic acid (Figure 3). The resulting renatured SLURP-1 preparation was lyophilized.

The described isolation method allows to obtain 1.4 mg of renatured SLURP-1 from 1 g of wet biomass, which corresponds to 1.4 mg of protein from 100 mg of lyophilized biomass.

The spectra measured on an UltraFlex TOF / TOF time-of-flight mass spectrometer (Bruker Daltonics, Germany) equipped with an MALDI ionization source proved the correspondence of the molecular weight of recombinant SLURP-1 to the calculated data obtained for the amino acid sequence of SEQ No. 8.

Example 6. Analysis of the secondary structure of recombinant renatured SLURP-1 by circular dichroism spectroscopy.

The CD spectrum is measured using a J-810 spectropolarimeter (Jasco, Japan) in a cuvette with an optical path length of 0.01 cm in the wavelength range of 190-250 nm in increments of 1 nm. The conformation of SLURP-1 in an aqueous solution (pH ~ 4.0) is examined. The concentration of SLURP-1 in water is 0.09 mm. Evaluation of the content of elements of the secondary structure (see table) is carried out according to the CONTINLL program [Provencher, S.W., Glocker, J. Estimation of globular protein secondary structure from circular dichroism. // Biochemistry. - 1982. - Vol.20. - P.33-37].

An analysis of the obtained spectrum shows that in an aqueous medium the recombinant renatured SLURP-1 has an ordered structure of the type of β-folds (table). The content of the β-structure is close to the structure observed in structural homologues of SLURP-1, snake neurotoxins.

Table Data on the secondary structure of recombinant renatured SLURP-1 obtained using circular dichroism The content of α-structure Β-layer content The content of β-turns Disordered content SLURP-1 four% 39% 24% 33%

Thus, the claimed technical solution allows to obtain a protein with a structure identical to the structure of natural human SLURP-1, and the level of its bacterial synthesis is at least 10% of the total cellular protein. This allows to obtain recombinant SLURP-1 in milligram quantities, which can be used in medicine as a neuromodulator for the treatment of diseases of the nervous system, as well as for the treatment of psoriasis and cancer.

Claims (2)

1. Recombinant plasmid DNA pET22b (+) / SLURP-1 encoding a 5660 bp human SLURP-1 protein, consisting of NdeI / BamHI, a pET22b (+) plasmid DNA fragment of 5403 bp in length containing the lac promoter E. coli, β-lactamase bla gene, E. coli lacI gene fragment, replication initiation ori region and artificial DNA sequence encoding a 257 bp NdeI / BamHI fragment including the synthetic human SLURP-1 gene as SEQ ID NO : 7; containing unique recognition sites by restriction endonucleases having the following coordinates: XhoI - 158, HindIII - 173, EcoRI - 192, BamHI - 198, NdeI - 430, BglII - 534, MluI - 1256, PstI - 4495. 2. The bacterial strain Escherichia coli BL21 (DE3) / pET22b (+) / SLURP-1 is a producer of human SLURP-1 protein obtained by transformation of competent Escherichia coli BL21 (DE3) cells with recombinant plasmid DNA pET22b (+) / SLURP-1.

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