RU2571942C2 - Method for obtaining recombinant analgesic peptide - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: inventions relate to vector construction, Escherichia coli strain, including such vector construction, and method for obtaining recombinant analgesic peptide PT1. Vector construction is obtained on the basis of vector pTWIN1 and contains gene of chimeric protein DnaB-PT1, which consists of chitin-binding domain from Bacillus circulans, intein DnaB from Synechocystis sp. and analgesic peptide PT1. Characterised method for obtaining includes carrying out controlled expression of gene of chimeric protein DnaB-PT1 in composition of characterised vector construction in E. coli strain-producent ER2566 with application of isopropyl-β-D-1-thiogalactopyranoside as inductor. After that lysis of cells, separation of soluble components, purification of chimeric protein DnaB-PT1 by means of affinity chromatography on chitin sorbent, autocatalytic cleavage of chimeric protein DnaB-PT1 and purification of peptide PT1 by means of reverse phase chromatography are performed.

EFFECT: claimed group of inventions makes it possible to obtain analgesic peptide with high output for application for medical purposes.

3 cl, 8 dwg, 6 ex

Description

The invention relates to biochemistry, specifically to biologically active polypeptides that have a modulating effect on cellular receptors involved in the generation of a pain signal and can find application in medicine and scientific research.

The invention solves the problem of producing a recombinant PT1 peptide that modulates the activity of P2X3 purinergic receptors and exhibits analgesic activity in animal models.

PT1 has the following amino acid sequence:

Gly ¹ -Tyr ² -Cys ³ -Ala ⁴ -Glu ⁵ -Lys ⁶ -Gly ⁷ -Ile ⁸ -Arg ⁹ -Cys ¹⁰ -Asp ¹¹ -Asp ¹² -Ile ¹³ -His ¹⁴ -Cys ¹⁵ -Cys ¹⁶ -Thr ¹⁷ -Gly ¹⁸ -Leu ¹⁹ -Lys ²⁰ -Cus ²¹ -Cys ²³ -Asn ²⁴ -Ala ²⁵ -Ser ²⁶ -Gly ²⁷ -Tyr ²⁸ -Asn ²⁹ -Cus ³⁰ -Val ³¹ -Cys ³² -Arg ³³ -Lys ³⁴ -Lys ³⁵

The PT1 peptide consists of 35 amino acid residues, can be isolated by chromatographic methods from the spider venom Alopecosa marikovskyi (formerly referred to as Geolycosa sp.) Or obtained using peptide synthesis as well as genetic engineering methods [Grishin EV et al. Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain // Ann. Neurol., 2010, Vol. 67, pp. 680-683; RF patent No. 2422459]. The PT1 peptide is capable of selectively inhibiting currents mediated by P2X3 purinergic receptors in rat sensory neurons, as well as under the conditions of expression of human P2X3 receptors in HEK 293 cells. It has been shown that, upon the action of nanomolar concentrations of the peptide, the activating effect of the natural ATP agonist is significantly reduced, which is associated with - apparently, with the stabilization of the desensitized state of the receptors. The concentration of PT1 causing 50% inhibition of currents mediated by P2X3 receptors in rat sensory neurons (IC ₅₀ ) is 12 ± 2 nM with a Hill coefficient (n _H ) of 1.1 ± 0.2. Studies of the activity of PT1 in relation to a number of ion channels and ionotropic receptors confirm the specificity of the action of the peptide on P2X3 receptors.

PT1-based drugs may be effective in the treatment of pain of various etiologies. Currently, there is a wide variety of low molecular weight compounds that modify the activity of P2X3 receptors [Andreev Y.A. et al. Molecules to selectively target receptors for treatment of pain and neurogenic inflammation // Recent Pat. Inflamm. Allergy Drag Discov., 2012, Vol. 6, pp. 35-45]. PT1 is the first compound of a polypeptide nature capable of exerting a selective modulating effect on P2X3 receptors. The peptide is characterized by an extremely stable structure (contains the motive of the so-called "cystine node"). This is the basis for the assumption of a high potential of PT1 in terms of drug development. The recently discovered peptide PT2 has properties similar to PT1 [Kabanova N.V. et al. Modulation of P2X3 receptors by spider toxins // Biochim. Biophys. Acta, 2012, Vol. 1818, pp. 2868-2875], however, significantly larger sizes, which makes it difficult to obtain in quantities necessary for medical purposes.

Most of the therapeutic polypeptides on the market are recombinant [Dimitrov D.S. Therapeutic proteins // Methods Mol. Biol., 2012, Vol. 899, pp. 1-26]. And about 30% of them are obtained in Escherichia coli [Huang C.J. et al. Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements // J. Ind. Microbiol. BiotechnoL, 2012, Vol. 39, pp. 383-399]. Recombinant PT1 was also obtained in E. coli in 3 mg yield with 1 L of bacterial culture [RF patent No. 2422459]. For this, the gene encoding PT1 was cloned into the expression vector pET-32b (Novagen, USA). The desired peptide was obtained in the form of a chimera with a helper protein (thioredoxin). The separation of the PT1 peptide from thioredoxin was carried out by hydrolysis of a chimera with the catalytic subunit of human enterokinase. This prototype method of the claimed is easily implemented and in demand on a research laboratory scale, but it is too expensive and inconvenient to scale.

The task of obtaining the peptide PT1 is solved due to the fact that the gene for the analgesic peptide PT1 is fused to a sequence encoding a chitin-binding domain from Bacillus circulans [Chong S. et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element // Gene, 1997, Vol. 192, pp. 271-281] and intein DnaB from Synechocystis sp. [Yan S.S. et al. Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli II Protein Expr. Purif., 2005, Vol. 40, pp. 340-345]. This allows the purification of the chimeric protein using affinity chromatography on chitin, and also to separate the desired recombinant PT1 from auxiliary elements using autocatalytic cleavage of the chimeric protein. The specified method allows to obtain the PT1 analgesic peptide with a high yield for medical use and eliminates the disadvantages of the prototype.

The method is as follows:

1) the sequence encoding the PT1 analgesic peptide is cloned into the pTWIN1 vector (New England Biolabs, USA), whereby the vector construct pER-PT1 is obtained,

2) the vector construct pER-PT1 is used to transform E. coli strain ER2566, resulting in a producing strain of E. coli ER2566 / pER-PT1,

3) carry out the controlled expression of the chimeric gene,

4) isolate the chimeric protein DnaB-PT1, consisting of a chitin-binding domain, intein DnaB and peptide PT1, using affinity chromatography on a chitin sorbent,

5) conduct autocatalytic cleavage of the fusion protein DnaB-PT1,

6) purify recombinant PT1 using reverse phase chromatography.

The technical result of the invention consists in:

1) obtaining a chimeric protein DnaB-PT1 containing a chitin-binding domain, intein DnaB and peptide PT1, due to the expression of the chimeric gene in the vector construct pER-PT1 used to create the producer strain E. coli ER2566 / pER-PT1;

2) obtaining recombinant PT1 due to autocatalytic cleavage of the fused protein DnaB-PT1.

The invention is illustrated by graphic materials:

Figure 1. A) The nucleotide sequence encoding the PT1 peptide. B) Primers used in amplification. The restriction sites KpnI, BamHI and SapI are underlined.

Figure 2. Map of the expression vector pER-PT1. DnaB- DnaB gene of mini-intein from Synechocystis sp. PT1 - PT1 gene.

Figure 3. Amino acid sequence of the DnaB-PT1 fusion protein.

Figure 4. Analysis of the productivity of the producer strain PT1. Electrophoresis in 15% SDS page under denaturing conditions. 1 - total cell lysate of E. coli ER2566 / pER-PT1; 2 - molecular weight standards.

Figure 5. Selection of culture conditions for producer E. coli ER2566 / pER-PTl. Electrophoresis in 15% SDS page under denaturing conditions. 1, 4, 7 - cultivation at 20 ° C for 2, 3 and 4 hours after the addition of the inductor; 2, 5, 8 - cultivation at 30 ° C for 2, 3 and 4 hours after the addition of the inductor; 3, 6, 9 - cultivation at 37 ° for 2, 3 and 4 hours after the addition of the inductor; 10 - molecular weight standards.

Figure 6. Analysis of autocatalytic cleavage of the DnaB-PT1 fusion protein. Electrophoresis in 15% SDS page under denaturing conditions. 1 - molecular weight standards; 2 - total cell lysate after 4 hours of cultivation of the producer from the moment of addition of the inducer; 3 - fraction not adsorbed on an affinity sorbent; 4 - a hybrid protein adsorbed on chitin; 5 - hybrid protein after 4 hours of incubation; 6 - soluble fraction of the hybrid protein after 12 hours of incubation; 7 - hybrid protein adsorbed on chitin after 12 hours of incubation; A - DnaB-PT1; B - DnaB, C - PT1.

Figure 7. Chromatography-mass spectrometric analysis of the pooled fraction obtained after affinity chromatography and containing PT1. An 6224 ESI-TOF device (Agilent, USA) with a YMC-Pack C ₈ column (150 × 2.1 mm; YMC Europe, Germany) was used. I is the chromatogram; II - mass spectrum of peak A.

Figure 8. Purification of PT1 using reverse phase chromatography on a Diasorb 130 C16T column. Selected fractions containing PT1 are highlighted in gray.

The invention is illustrated by examples.

Example 1. The creation by genetic engineering of an expression vector containing a hybrid gene with an integrated sequence encoding PT1

The PT1 peptide gene (Figure 1A), modulating the activity of P2X3 purinergic receptors, is amplified by PCR (according to the program: 92 ° C - 1 min, then 30 cycles: 92 ° C - 30 s, 60 ° C - 30 s, 72 ° C - 30 c) with plasmid DNA pET-32b-PT1 [RF patent No. 2422459] as a template and PTIN-1 and PTEV-2 oligonucleotides (Figure 1B) as primers.

The amplifications are treated with restriction endonucleases SapI, BamHI and ligated with the vector pTWIN1, also pretreated with the aforementioned endonucleases. The competent E. coli ER2566 cells are transformed with the ligase mixture, which are then grown on YT agar medium containing ampicillin at a concentration of 100 μg / ml overnight (16 hours) at 37 ° C. The selection of transformants is carried out by analysis using electrophoresis in 15% polyacrylamide gel (PAAG) under denaturing conditions. The result is a recombinant vector pER-PT1 (Figure 2), carrying the DnaB-PT1 fusion protein gene sequence shown in Figure 3. When expressing a hybrid gene encoding an auxiliary DnaB mini-intein, a fusion protein capable of autocatalytic under certain conditions should be formed cleavage and release of the target peptide PT1.

E. coli ER2566 cells are transformed with the obtained recombinant vector, and E. coli producer strain ER2566 / pER-PT1 is obtained.

Example 2. Evaluation of the expression level of the hybrid gene encoding PT1 in the obtained producer strain

YT medium containing 100 μg / ml ampicillin is seeded with overnight culture (1%) of E. coli strain ER2566 / pER-PT1. The cultivation is carried out in flasks at 37 ° C until the optical absorption of the culture medium OD ₆₀₀ = 0.6. Isopropyl-β-D-1-thiogalactopyranoside inducer (IPTG; Sigma, USA) was added to a final concentration of 0.4 mM, the culture was incubated for 4 hours at 37 ° C. With 1 l of culture, 5 g of wet cells are obtained. Productivity is assessed by analysis by electrophoresis in 15% SDS page under denaturing conditions (Figure 4). Significant autocatalytic cleavage of the fusion protein in vivo is observed for E. coli strain ER2566 / pER-PT1.

Example 3. Conditions for the expression of a chimeric gene and isolation of the target product

An important step is to test the ability of the obtained fusion protein containing intein to autocatalytic cleavage and release of the target product.

At the stage of evaluating the effectiveness of the obtained producer strain, it was noted that under standard cultivation conditions, the hybrid protein undergoes autocatalytic cleavage in vivo by approximately 50% (Figure 4). Therefore, at first, optimization of the growth conditions of the culture is carried out, the temperature and time of cultivation are varied (Figure 5). The optimal conditions are: the addition of an inductor at OD ₆₀₀ = 2 and further cultivation at 30 ° C for 2.5 hours. In this case, the hybrid protein undergoes autocatalytic cleavage in vivo by 15-20%.

YT medium containing 100 μg / ml ampicillin is seeded with overnight culture (1%) of E. coli strain ER2566 / pER-PT1. The cultivation is carried out in flasks at 37 ° C until the optical absorption of the culture medium OD ₆₀₀ = 2. An IPTG inducer was added to a final concentration of 0.4 mM, the culture was incubated for 2.5 hours at 30 ° C. With 1 l of culture, 5 g of wet cells are obtained.

Cells (100 mg) are resuspended in 2 ml of buffer A (50 mm Tris-HCl, pH 8.5, 50 mm EDTA, 1 mm PMSF) and then lysed using an ultrasonic disintegrator. The cell lysate is centrifuged, the supernatant is separated from the precipitated components, diluted 2 times with buffer B (50 mm Tris-HCl, pH 8.5, 1 mm EDTA, 1 mm PMSF). 4 ml of the obtained solution of the target hybrid protein is applied to a chromatographic column containing 1 ml of chitin sorbent and previously equilibrated with buffer B. Column washing is carried out with 10 ml of buffer B, after which 3 ml of buffer B (50 mM Tris-HCl, pH 6.5, 1 mM EDTA, 1 mM PMSF). The column was incubated for 16 hours at 30 ° C. It turns out that the degree of cleavage of the hybrid protein reaches ~ 100% (Figure 6).

The PT1 peptide cleaved from the fusion protein is eluted with 1 ml of buffer B and analyzed by reverse phase high performance liquid chromatography (RP-HPLC) (Figure 7). The analysis established that, along with the target product (A in Figure 7), the eluate contains products of incomplete oxidation and a by-product with a molecular weight identical to PT1 (B in Figure 7). Product A was purified by RP-HPLC on a Macrosphere RP 300 Cig column (250 x 4.6 mm; Alltech, USA). Mass spectrometric analysis confirms that product A corresponds to PT1.

The fractions obtained after affinity chromatography and containing the target peptide are combined, applied to a Macrosphere RP 300 C ₁₈ column, previously equilibrated with solution G (5% acetonitrile, 0.1% trifluoroacetic acid). Chromatographic separation is carried out in a linear gradient of acetonitrile concentration (5-40%) for 25 minutes at a rate of 0.7 ml / min. Fractions containing the desired PT1 peptide with a purity of not less than 98% are combined and freeze-dried. The output of PT1 is 12 mg with 1 l of bacterial culture.

The main drawback in the protocol is the autocatalytic cleavage of the DnaB-PT1 fusion protein in vivo, however, the optimization of cultivation conditions can significantly reduce the degree of cleavage (up to 10-15%). Thus, the isolation protocol using the E. coli producer strain ER2566 / pER-PT1 is effective.

Example 4. The optimized Protocol for the preparation of recombinant peptide analgesic PT1 using producer strain E. coli ER2566 / pER-PT1

20 g of producer cells are resuspended in 150 ml of buffer A and then lysed using an ultrasonic disintegrator (20 s pulse / 20 s cooling, 20 cycles). The cell lysate is centrifuged at 12,000 g for 30 minutes. The supernatant is separated from the precipitated components, diluted 2 times with buffer B. 300 ml of the obtained hybrid protein solution is applied to a chromatographic column containing 50 ml of chitin sorbent and previously balanced with buffer B. The column is washed with 200 ml of buffer B, after which 100 ml of buffer B is fed. The column is incubated for 16 hours at 30 ° C. The degree of cleavage of the hybrid protein is ~ 100%. The PT1 peptide cleaved from the fusion protein is eluted with 80 ml of buffer B. The fractions containing PT1 are combined.

Then the PT1 solution was applied to a Diasorb 130 C16T chromatographic column (250 × 16 mm; Elsiko, Russia), previously equilibrated with solution G, at a rate of 1.5 ml / min. Separation is carried out in a linear gradient of acetonitrile concentration (5-40%) for 80 min at a rate of 3 ml / min (Figure 8). Fractions containing peptide PT1 with a purity of not less than 98% are combined and freeze-dried. The yield is 57 mg per 20 g of wet cells (14.3 mg per 1 liter of culture).

Example 5. The production and analysis of prototypes PT1 on the content of impurities

The production of PT1 is carried out in accordance with the developed optimized protocol using the created E. coli producer strain ER2566 / pER-PT1.

Analysis of the prototypes carried out in the control and analytical laboratory of the Institute of Bioorganic Chemistry, Russian Academy of Sciences, shows that the preparation obtained is safe and meets pharmacopoeial requirements in terms of “Bacterial endotoxins”, “Residual DNA of producer strain” and “Level of bacterial endotoxins”.

The amino acid sequence of recombinant PT1 is determined using automatic sequencing according to Edman. It corresponds to the natural: GYCAEKGIRCDDIHCCTGLKCKCNASGYNCVCRKK. The molecular weight of recombinant PT1 is measured using mass spectrometry on an Ultraflex II instrument (Bruker, Germany). It is 3833.6 Yes and is equal to the calculated value.

Example 6. Determination of the biological activity of recombinant PT1

The resulting PT1 preparation is tested on recombinant human P2X3 receptors. For this, the human P2X3 gene, cloned into the pIRES2-EGFP vector (Clonteth), is used to transfect HEK 293 cells. Transfection is carried out according to the standard method using lipofectamine (Invitrogen, USA). Electrophysiological testing is carried out using the classical method of local fixation of potential ("patch clamp") in the configuration of voltage removal from the whole cell ("whole cell") using the approach of rapid application of the agonist. Currents are recorded at room temperature using a Model 2400 amplifier (AM Systems, USA) with a supported potential of -60 mV. As agonists that cause P2X3-mediated currents, adenosine triphosphate (ATP) and cytidine triphosphate (CTF) are used. The application of PT1 to receptors in an insensitive (desensitized) state leads to a significant decrease in the amplitude of the current upon subsequent activation. The experimental data are analyzed according to the Hill model. The concentration of PT1, at which a two-fold decrease in the amplitude of the current is observed compared with the control value, (IC ₅₀ ) is 20 nM.

Thus, the claimed method allows to obtain a recombinant peptide analgesic PT1, fully consistent with the native peptide in structure and biological activity.

Claims (3)

1. Vector construct based on the vector pTWIN1 and containing the gene for the chimeric protein DnaB-PT1, which consists of a chitin-binding domain from Bacillus circulans, intein DnaB from Synechocystis sp. and the PT1 analgesic peptide, and allowing for controlled expression of the fusion gene. 2. The strain Escherichia coli is a producer of the chimeric protein DnaB-PT1, which is a strain of E. coli ER2566 with an integrated vector construct according to claim 1. 3. A method of producing a recombinant analgesic peptide PT1, including the controlled expression of the gene for the chimeric protein DnaB-PT1 as part of the vector construct according to claim 1 in the E. coli producer strain according to claim 2 using isopropyl-β-D-1 as an inducer -thiogalactopyranoside, cell lysis, separation of soluble components, purification of the chimeric protein DnaB-PT1 using affinity chromatography on a chitin sorbent, autocatalytic cleavage of the chimeric protein DnaB-PT1, purification of the peptide PT1 using reverse phase chromatography.

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