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RU-2861683-C2 - METHOD FOR PRODUCING RECOMBINANT HUMAN SOMATOTROPIN

RU2861683C2RU 2861683 C2RU2861683 C2RU 2861683C2RU-2861683-C2

Abstract

FIELD: biotechnology. SUBSTANCE: new method for producing recombinant human somatotropin is proposed, including fermentation with a genome built into the E. coli BL21(DE3) chromosome, production of a hybrid somatotropin protein with an N-terminal partner protein, and subsequent purification using the described method with isolation of the target product. EFFECT: to ensure high purity and high yield of human somatotropin with the correct N-terminal sequence. 5 cl, 3 dwg, 1 tbl, 3 ex

Inventors

  • KARASEV VIKTOR SEMENOVICH
  • Bochkova Olga Petrovna
  • STAROVEROV SERGEJ MIKHAJLOVICH
  • SHULGA ALEKSEJ ANATOLEVICH

Dates

Publication Date
20260507
Application Date
20211130

Claims (16)

  1. 1. A method for producing recombinant human somatotropin, comprising
  2. carrying out fermentation with a gene integrated into the E. coli BL21(DE3) chromosome, which ensures the production of a hybrid protein of somatotropin with an N-terminal partner protein,
  3. destruction of biomass,
  4. preparation of ammonium sulfate precipitate,
  5. its dissolution in a buffer solution containing sodium phosphate, sodium chloride and imidazole, with the isolation from the solution of the hybrid protein somatotropin with the N-terminal partner protein and its purification by sequentially implementing the following steps:
  6. a) metal-chelate chromatography on a sorbent with iminodiacetic acid in nickel form;
  7. b) removing salts from the eluate obtained in stage a) by passing the eluate through a hydrophilic sorbent to obtain a desalted eluate;
  8. c) enzymatic cleavage of the hybrid protein contained in the desalted eluate from step b) with proteinase, ensuring the production of somatotropin with the correct N-terminal sequence;
  9. d) removing impurities from the solution obtained in stage c) by carrying out metal chelate chromatography on a sorbent with iminodiacetic acid in nickel form with collection of fractions of the solution containing the target product;
  10. d) chromatographic purification of the solution obtained in stage d) using a strongly basic anion exchanger;
  11. e) concentrating the purified solution obtained in step d) containing the target product using a strongly basic anion exchanger;
  12. g) gel filtration chromatography by passing the concentrated solution obtained in step e) through a column filled with a hydrophilic sorbent to replace the buffer medium.
  13. 2. The method according to paragraph 1, characterized in that the hydrophilic sorbent used is a sorbent selected from the series Sephadex G-25 (GE), WorkBeads DSalt (Bio Works) and BioGel (BioRad).
  14. 3. The method according to claim 1, characterized in that for metal-chelate chromatography, sorbents selected from the series are used: Ni-IDA Sepharose 6B (GE), WorkBeads 40 IDA in nickel form (Bio Works) and ReliSorb IDA400 (Resindion).
  15. 4. The method according to paragraph 1, characterized in that the anion exchanger used is anion exchange material from the series: MCI-Gel, Q-Sepharose FF and Toyoperl Super Q.
  16. 5. The method according to claim 1, characterized in that the enzymatic cleavage of the hybrid protein is carried out at a mass ratio of hybrid protein:proteinase of 1:10000 (w/w) ensuring the correct N-terminal sequence of somatotropin.

Description

The invention relates to the field of biotechnology, in particular to a method for isolating and purifying recombinant human growth hormone (rHGH), the so-called somatotropin (somatropin). A known method for obtaining and purifying rHGH involves dissolving inclusion bodies in 2 M urea at pH 11.0 and renaturing the protein in a 20 mM Tris-HCl buffer at pH 8.0. Hydrogen peroxide is used as an oxidizer for the sulfhydryl groups of rHGH. The N-terminal methionine is cleaved using leucine aminopeptidase. Purification of rHGH is carried out using two-stage ion-exchange chromatography on Q Sepharose FF sorbent and hydrophobic interaction chromatography on Butyl Sepharose 4 FF sorbent. (RU 2473556, 2013). The known method is based on the renaturation of inclusion bodies in a soluble active hormone, which results in significant losses of the target product. The yield of the finished product is approximately 45%. Furthermore, the resulting product is prone to aggregation, which reduces its shelf life. A method for isolating and purifying recombinant human growth hormone secreted by Saccharomyces cerevisiae yeast during fermentation under appropriate conditions is known. According to the method, the target protein is precipitated from the biomass-free culture fluid by acidification to a pH of 2.9-4.0 or by adding 3000-6000 Da of polyethylene glycol. The resulting precipitate is dissolved in a suitable solvent. The target protein is purified either by anion-exchange chromatography at a pH of 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. The primary purification of the target protein is then carried out by anion-exchange chromatography at a pH of at least 7.3 and gel filtration. The invention enables the production of growth hormone free of related proteins, host proteins, and other impurities, such as pigments. The yield of the product, according to this method, chosen as a prototype, is up to 60% (RU 2492177, 2013). A disadvantage of this known method is the low yield of the target product, as the use of yeast does not allow for the secretion of the required amount of protein. Furthermore, glycosylation may occur in the medium used, which may lead to a decrease in the biological activity of the product. The aim of the present invention is to develop a new method for producing recombinant human somatotropin, ensuring high purity and high yield of the target product, as well as expanding the range of industrial methods for producing this product. The stated problem is solved by the described method for obtaining recombinant human somatotropin, which includes fermentation with a gene integrated into the chromosome of E. coli BL21(DE3), which ensures the production of a hybrid protein of somatotropin with an N-terminal partner protein, destruction of the biomass, preparation of an ammonium sulfate precipitate, its dissolution in a buffer solution containing sodium phosphate, sodium chloride and imidazole, with the isolation from the solution of the hybrid protein of somatotropin with an N-terminal partner protein and its purification by sequentially implementing the following stages: a) metal-chelate chromatography on a sorbent with iminodiacetic acid in nickel form; b) removing salts from the eluate obtained in stage a) by passing the eluate through a hydrophilic sorbent to obtain a desalted eluate; c) enzymatic cleavage of the hybrid protein contained in the desalted eluate from step b) with proteinase, ensuring the production of somatotropin with the correct N-terminal sequence; d) removal of impurities from the solution obtained in stage c) by carrying out metal chelate chromatography on a sorbent with iminodiacetic acid in nickel form with collection of fractions of the solution containing the target product; d) chromatographic purification of the solution obtained in stage d) using a strongly basic anion exchanger; e) concentrating the purified solution obtained in step d) containing the target product using a strongly basic anion exchanger; g) gel filtration chromatography, by passing the concentrated solution obtained in step e) through a column filled with a hydrophilic sorbent to replace the buffer medium. Preferably, sorbents selected from the series Sephadex G-25 (GE), WorkBeads DSalt (BioWorks) and BioGel (BioRad) are used as a hydrophilic sorbent. Preferably, for metal chelate chromatography, sorbents selected from the following range are used: WorkBeads 40 IDA in nickel form, Ni-IDA Sepharose 6B and Resindion IDA-400. Preferably, anion exchange materials from the following series are used as anion exchangers: MCI-GELCPQ10/P60 (Mitsubishi, Japan), Nuvia HP-Q (BioRad), Q-Sepharose FF(GE) or Toyoperl Super Q (Tosoh). Preferably, the cleavage of the hybrid protein is carried out at a ratio of hybrid protein:proteinase of 1:10000 (w/w), respectively, to achieve the correct N-terminal sequence of somatotropin. The claimed production method, as defined in the independent claim, yields a sol