RU2714114C1 - Method of producing a peptide which modulates purinergic receptor activity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of biologically active polypeptides, which have modulating action on involved in generation of pain signal cell receptors, and can be used to produce peptide PT6 in an expression system of E. coli.

EFFECT: invention provides high-efficiency production of peptide PT6, which modulates purinergic receptor activity, in Escherichia coli BL21 (DE3) cells with high output.

1 cl, 7 dwg

Description

Technical field

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, and a method for their preparation.

State of the art

The study and treatment of pain are essential aspects of improving the quality of life of patients. Pain treatment is mainly based on the use of anti-inflammatory drugs, which can be non-steroidal anti-inflammatory drugs (NSAIDs) or steroidal (corticosteroid) drugs, antidepressants, anticonvulsants, and also strong or weak opiates. Despite the large number of existing therapeutic agents, many types of pain remain insensitive to known analgesics, therefore, the development of new analgesic agents with a new mechanism of action, as well as the development of effective methods for their preparation, are an important and urgent task.

The PT6 peptide modulating purinergic signaling [RU2650780] is known in the art. Along with this, methods are known for producing a PT1 peptide (homologous) close in structure to PT6, consisting of 35 amino acid residues and modulating the activity of P2X3 purinergic receptors. PT1 can be obtained in a bacterial expression system in the laboratory [Grishin EV et al. Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain // Ann. Neurol., 2010, Vol. 67, p. 680-683; RU2422459; RU2571942]. A known industrial method for producing PT1 ["Method for producing recombinant analgesic peptide", patent RU2571942]. It consists in the production in the Escherichia coli strain ER2566 of the chimeric protein DnaB-PT1, which consists of a chitin-binding domain from Bacillus circulans , an intein DnaB from Synechocystis sp. and the PT1 analgesic peptide, purification of the DnaB-PT1 chimeric protein by affinity chromatography on a chitin sorbent, autocatalytic cleavage of the DnaB-PT1 chimeric protein, and purification of the PT1 peptide by reverse phase chromatography. The disadvantage of this method is the relatively low yield of the target product (14.3 mg per 1 liter of culture).

The closest analogue of the claimed invention is a laboratory method for producing the PT6 peptide described in RU2650780. It consists in the production in the E. coli strain BL21 (DE3) of the chimeric protein Trx-PT6, which consists of the helper protein thioredoxin and the peptide PT6, purification of the chimeric protein Trx-PT6 using affinity chromatography on sorbent TALON Superflow Metal Affinity Resin (Clontech) cleavage of the chimeric Trx-PT6 protein with enteropeptidase; and purification of the PT6 peptide using reverse phase chromatography. The disadvantage of this method is the low yield of the target product (5 mg with 1 l of culture). Despite the fact that there are methods for producing compounds modulating the activity of purinergic receptors, the effectiveness of these methods is rather low.

Disclosure of Invention

The present invention is to develop a new effective method for producing peptide PT6. The PT6 peptide consists of 31 amino acid residues and has the amino acid sequence of SEQ ID NO 1.

The technical result consists in the development of a new effective method for producing the PT6 peptide, which allows one to obtain a peptide with a high yield of the target product of 82.5 mg per 1 liter of culture, the specified yield being several times higher than with the already known methods for producing such a peptide.

In addition, the technical result of the invention consists in:

1) obtaining the chimeric protein Trx-PT6 (SEQ ID NO 2) containing the helper protein thioredoxin and peptide PT6, due to the expression of the chimeric gene in the vector construct pET-32b-PT6 used to create the producer strain E. coli BL21 (DE3 ) / pET-32b-PT6;

2) obtaining recombinant PT6 due to cleavage of the chimeric protein Trx-PT1 by enteropeptidase.

The specified technical result is achieved through the development and implementation of a method for producing peptide PT6 with the sequence of SEQ ID NO 1, including:

- conducting controlled expression of the gene for the chimeric protein Trx-PT6 with the sequence SEQ ID NO 2 in the vector construct based on the vector pET-32b (Novagen), and this design contains the gene for the chimeric protein Trx-PT6 and allows for the controlled expression of the fusion gene in the strain producer E. coli BL21 (DE3) with an integrated vector construction using isopropyl-β-D-1-thiogalactopyranoside as an inductor,

 - the destruction of the cells of the producer strain with the formation of a lysate,

 - separation of the soluble components of the obtained lysate,

 - purification of the chimeric protein Trx-PT6 using ion exchange chromatography,

 - cleavage of the chimeric protein Trx-PT6 enteropeptidase,

 - purification of the peptide PT6 using ion exchange and reverse phase chromatography.

Detailed Disclosure of Invention

Brief Description of the Drawings

Figure 1 . Electrophoretic analysis of the total cell lysate obtained after the destruction of E. coli BL21 (DE3) / pET-32b-PT6 cells. 1 - before induction of fusion protein gene expression, 2 - 2 hours after induction, 3 - after the end of the fermentation process, 4 - molecular weight standards (Thermo Scientific Unstained Protein Molecular Weight Marker), the mass of standard proteins in kDa is indicated on the right. 10 μg of protein was applied to the tracks.

Figure 2. Chromatographic profile of Trx-PT6 fusion protein purification on a DEAE Sepharose Fast Flow anion-exchange sorbent. Fractions containing over 60% of the target component are marked with a fill.

Figure 3. Electrophoretic analysis of fractions obtained by purification of the Trx-PT6 fusion protein on the DEAE Sepharose Fast Flow anion exchange sorbent. Electrophoresis was performed on a 14% polyacrylamide gel under denaturing conditions. 1 - sample before separation on a sorbent DEAE Sepharose Fast Flow, 32%; 2 - fraction 2 (figure 2), 49%; 3 - fraction 3, 72%; 4 - fraction 4, 79%; 5 - fraction 5, 57%; 6 - fraction 6; 7 — fraction 7. 10 μg of protein was applied to the tracks. These percentages correspond to the content of the Trx-PT6 fusion protein.

Figure 4. The chromatographic profile of the purification of the peptide PT6 on the anion-exchange sorbent SP Sepharose Fast Flow. Fractions containing over 98% of the target component are marked with a fill.

Figure 5. The chromatographic profile of the purification of the peptide PT6 on a reversed-phase sorbent YMC Basic. A fraction containing PT6 with a purity of at least 98% is marked with a fill.

Figure 6. Chromatographic profile of the purification of the PT6 peptide by gel filtration on a Sephadex G-25 Superfine sorbent. A fraction containing PT6 with a purity of at least 98% is marked with a fill.

Figure 7. Chromatographic analysis of the lyophilisate of the RT6 peptide by RP-HPLC. VDSpher PUR 100 C18-E column (4.6 × 250 mm; particle size 5 μm; VDS optilab Chromatographietechnik). A gradient separation of acetonitrile (from 9.6 to 24% in 20 minutes) in 0.1% trifluoroacetic acid with an elution rate of 1 ml / min.

Definitions and Terms

Various terms related to the objects of the present invention are used above and also in the description and in the claims. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as is understood by specialists in this field. References to the techniques used in the description of this invention relate to well-known methods, including modifying these methods and replacing them with equivalent methods known in the art.

In the description of the present invention, the terms “ includes ” and “ including ” are interpreted as meaning “includes, among other things.” These terms are not intended to be construed as “consists of only”.

As used herein, the term " PT6 peptide " refers to a peptide having the following amino acid sequence:

The term “ modulate ” as used herein means changing the functional characteristics of P2X purinergic receptors (in particular, inhibiting, blocking, decreasing, or preventing physiological effects caused by the binding of an agonist, including an endogenous agonist, to a receptor), in particular P2X3 receptors.

The term “ modulator ” as used herein means a substance capable of modulating, that is, altering the functional characteristics of P2X purinergic receptors (in particular, inhibiting, blocking, decreasing or preventing physiological effects caused by binding of an agonist, including an endogenous agonist), in particular P2X3 receptors.

The implementation of the invention

The construction of recombinant plasmid DNA pET-32b-PT6 encoding the PT6 peptide is described in RU2650780. The E. coli BL21 (DE3) / pET-32b-PT6 producer strain is obtained by transformation of competent E. coli BL21 (DE3) cells with the plasmid pET-32b-PT6 (RU2650780). Inoculum in flasks is obtained by culturing the producer strain E. coli BL21 (DE3) / pET-32b-PT6 in a nutrient medium containing aminotone (10 g / l; A. Costantino & C. spa), yeast extract (5 g / l; Biospringer), NaCl (10 g / l), MgSO ₄ (0.25 g / l) and glucose (5 g / l), pH 7.0, with the addition of ampicillin (50 μg / ml). For this, 1 ml of thawed museum culture of the producer strain is introduced into 2 liters of culture medium, incubated for 16 hours at a temperature of 37 ° C and a stirring speed of 210 rpm.

Then the producer is grown in a fermenter. To do this, prepare 130 l of a nutrient medium containing aminotone (20 g / l), yeast extract (10 g / l), K ₂ HPO ₄ × 3H ₂ O (5 g / l), NaCl (4 g / l), MgSO ₄ (0.4 g / l) and glucose (5 g / l), which is loaded into a fermenter with a capacity of 0.3 m ³ (MBR), add 35 g of antifoam "Laprol" (Nizhnekamskneftekhim). The following technological parameters are set for the fermenter: a stirrer rotation speed of 30%, a supply of sterile air in an amount of 0.1 m ³ / min, an overpressure of 0.4 atm, a temperature of 37 ° C, a reading of a dissolved oxygen sensor (pO ₂ ) of 100%. A sterile ampicillin solution, the final concentration of which is 100 mg / l, is introduced into the fermenter by a peristaltic pump through the feed transfer line. The seed is connected to the fermenter, using a peristaltic pump, the producer culture is inoculated into the nutrient medium at the rate of 2 liters of seed per 130 liters of nutrient medium.

The process of growing producer cells is carried out, automatically maintaining the following parameters: temperature 37 ° C, pressure 0.4 atm, air flow 0.1 m ³ / min, mixing speed 90-270 rpm to maintain a pO ₂ value _of 30-90%, the concentration of dissolved oxygen pO ₂ 50-100%, pH 7.0. To adjust the pH during the growth of biomass by a peristaltic pump, a 50% glucose solution and a 25% ammonia solution are fed through a feed transfer line. After 5 hours from the time of culture inoculation of the producer in the fermenter with an optical density of the culture fluid A _{600 nm} = 7, the synthesis of the chimeric protein in the culture cells is carried out by introducing the peristaltic pump into the fermenter under aseptic conditions through the feed line of the isopropyl-β-D-thiogalactopyranoside solution, the final concentration of which is 50 mg / l. 4 hours after the introduction of the inducer of biosynthesis of the chimeric protein, the fermentation process is stopped. As a result, 135 L of producer cell culture fluid is obtained, which is fed to a CSA separator 19-06-476 (GEA Westfalia Separator), two unloadings of producer cell suspension with a total volume of 11.2 L from the separator are carried out. The figure 1 shows the electrophoretic analysis of samples of total cellular protein before induction of expression of the chimeric gene, as well as 2 hours after induction and after fermentation.

The resulting suspension is diluted to 57.8 L with water, Tris (total concentration 50 mM) and EDTA (total concentration 10 mM) are added. The destruction of biomass cells is carried out using an MC15 disintegrator (211, 10 TBSI; Gaulin) at a temperature of 13.5 ° C, a stirring speed of 400 rpm (40%) and a pressure of 590 atm for 20 minutes. For three cycles, 56.6 L of a suspension of destroyed cells is obtained.

Then, three consecutive cycles of obtaining the disintegrate supernatant are carried out on a CEPA Z81G flow-through centrifuge (Carl Padberg Zentrifugenbau) from the destroyed biomass (flow rate 20 l / h). For each cycle take 18.9 l of destroyed biomass of producer cells. At each sequential step, 18 L of disintegrate supernatant is obtained (54 L in total).

Isolation and purification of the Trx-PT6 fusion protein from E. coli BL21 (DE3) / pET-32b-PT6 cell lysate was performed by anion exchange chromatography on DEAE Sepharose Fast Flow anion exchange resin (GE Healthcare). Urea up to 4 M is added to the supernatant of the producer cell disintegrate (18 L; 50 mM Tris-HCl, pH 8, 10 mM EDTA), diluted 2.5 times with buffer A (50 mM Tris-HCl, pH 8.6), filtered diluted 4 times with buffer A and applied to a BPG 200 column (GE Healthcare) with anion exchange resin (4 L), balanced with buffer A. Separation is carried out at a speed of 20 l / h in a gradient mode from 0 to 100% buffer B (0, 2 M NaCl, 50 mM Tris-HCl, pH 8.6) per 32 L, fractions were collected by optical absorption of the eluate at 280 nm. Figure 2 shows the obtained separation profile, and figure 3 shows the result of electrophoretic analysis of the obtained fractions. The fractions containing over 60% of the target component are combined, resulting in a solution of purified Trx-PT6 fusion protein (15 L, 45 g). For three cleaning cycles, 45 L of a solution containing 135 g of Trx-PT6 fusion protein is obtained.

The Trx-PT6 fusion protein is digested with enteropeptidase. For this, the pH of the solution obtained by purification of the hybrid protein is adjusted to 8.5 using HCl, enteropeptidase (1 unit of enzyme per 1 mg of protein) is added to the solution, the mixture is incubated for 24 hours at room temperature with stirring. The result is a solution containing, according to estimates using reverse phase high performance liquid chromatography (RP-HPLC), 20 g of PT6.

Anion exchange chromatography was used to separate the desired PT6 peptide (pI ~ 10) and the byproduct of the thioredoxin helper protein (Trx; pI ~ 5.3). The Trx-PT6 fusion protein hydrolyzate is diluted 2 times with a solution of 50 mM Tris-HCl (pH 8.5) and a BPG 200 column is applied with a DEAE Sepharose Fast Flow sorbent at a rate of 20 l / h. The PT6 peptide is not retained on the sorbent and is contained in the slip in a volume of 90 l.

Further purification of the PT6 peptide is carried out using cation exchange chromatography. To this end, 18 L of a solution of 150 mM CH ₃ COONa was added to the slip from the previous step (anion exchange chromatography) and the pH was adjusted to 4.5 with glacial acetic acid with stirring. The resulting solution was applied to a BPG 200 column with SP Sepharose Fast Flow sorbent (2 L; GE Healthcare) equilibrated with buffer B (20 mM CH ₃ COONa, pH 4.5). Separation is carried out at a speed of 20 l / h in a gradient mode from 0 to 100% buffer G (0.5 M NaCl, 20 mm CH ₃ COONa, pH 4.5) per 40 l, the fractions are collected by optical absorption of the eluate at 280 nm. Figure 4 shows the obtained separation profile. Combine fractions containing more than 98% of the target component according to RP-HPLC. The result is 32 L of a solution containing 18.4 g of PT6 peptide.

For additional purification of the product from the residual amount of helper protein, RP-HPLC was used. Chromatographic purification is performed on a DAU-100-700 (YMC) column filled with a YMC Basic sorbent (15 μm, 20 nm; YMC Europe; 0.9 kg, 1.5 L). The PT6 peptide solution obtained in the previous step (cation exchange chromatography) was applied to a column equilibrated with solution A (0.3 M NaCl, 0.8% acetic acid) at a rate of 200 ml / min. After application, the sorbent is washed with 1.5 L of solution A. The target peptide is eluted with solution B (0.8% acetic acid) in isocratic mode, the fractions are collected by optical absorption of the eluate at 280 nm. Figure 5 shows the obtained separation profile. The result is an eluate in a volume of 2.2 l, containing 17.5 g of PT6 peptide with a purity of at least 98% according to analytical RP-HPLC.

At the final stage, the purification of the target PT6 peptide is performed by gel filtration on a BPG 200/950 column (GE Healthcare) using a Sephadex G-25 Superfine sorbent (5 kg, 25 L; GE Healthcare). The PT6 solution obtained in the previous step (RP-HPLC) was applied to a column equilibrated with a solution of 10 mM acetic acid at a rate of 10 l / h. Elution is carried out with the same solution, fractions are collected by optical absorption of the eluate at 280 nm. Figure 6 shows the obtained separation profile. The result is an eluate in a volume of 3.1 l, containing 16.9 g of PT6 with a purity of at least 98% according to analytical RP-HPLC.

At the final stage of the process, lyophilization of the purified substance of the PT6 peptide is performed. The solution obtained by gel filtration is passed through a sterile membrane filter with pores with a diameter of 0.22 μm (Millipore), poured into sterile drying trays and lyophilized. The result is a PT6 lyophilisate in an amount of 16.6 g with a moisture content of 7-8%, containing 15.4 g of PT6 according to analytical RP-HPLC. The figure 7 presents the chromatogram of the lyophilisate of the substance PT6.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the gist of the present invention.

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LIST OF SEQUENCES

<110> SOCIETY WITH LIMITED LIABILITY "ANALGETICS OF THE FUTURE"

<120> METHOD FOR PRODUCING A PEPTIDE MODULATING THE ACTIVITY OF PURINERGIC RECEPTORS

<130> 415042

<160> 2

<210> 1

<211> 31

<212> PRT

<213> Artificial sequence

<400> 1

Gly Tyr Cys Ala Thr Lys Gly Ile Lys Cys Asn Asp Ile His Cys Cys

  1      5             10          fifteen

Ser Gly Leu Lys Cys Asp Ser Lys Arg Lys Val Cys Val Lys Gly

          20          25        thirty

<210> 2

<211> 189

<212> PRT

<213> Artificial sequence

<400> 2

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

  1      5             10            fifteen

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

         20      25                 thirty

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

   35             40          45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

        fifty      55                 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

 65           70       75               80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

                              85                90            95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

                    100          105        110

Ser Gly His Met His His His His His His His Ser Ser Gly Leu Val Pro

             115           120       125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Gly Gln

       130       135   140

His Met Asp Ser Pro Asp Asp Leu Gly Thr Asp Asp Asp Asp Asp Arg Gly Tyr

145            150           155          160

Cys Ala Thr Lys Gly Ile Lys Cys Asn Asp Ile His Cys Cys Ser Gly

   165             170          175

Leu Lys Cys Asp Ser Lys Arg Lys Val Cys Val Lys Gly

         180         185

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Claims (7)

A method of obtaining a peptide PT6 with the sequence of SEQ ID NO: 1, including: - conducting controlled expression of the gene for the chimeric protein Trx-PT6 with the sequence SEQ ID NO: 2 in the vector construct based on the vector pET-32b, and this design contains the gene for the chimeric protein Trx-PT6 and allows for the controlled expression of the fusion gene in the producer strain E. coli BL21 (DE3) with embedded vector design, using isopropyl-β-D-1-thiogalactopyranoside as an inductor, - the destruction of the cells of the producer strain with the formation of a lysate, - separation of the soluble components of the obtained lysate, - purification of the chimeric protein Trx-PT6 using ion exchange chromatography, - cleavage of the chimeric protein Trx-PT6 enteropeptidase, - purification of the peptide PT6 using ion exchange and reverse phase chromatography.

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