WO2019070162A1 - Production strain of somatotropin, primarily in monomeric form - Google Patents

Production strain of somatotropin, primarily in monomeric form Download PDF

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WO2019070162A1
WO2019070162A1 PCT/RU2018/050040 RU2018050040W WO2019070162A1 WO 2019070162 A1 WO2019070162 A1 WO 2019070162A1 RU 2018050040 W RU2018050040 W RU 2018050040W WO 2019070162 A1 WO2019070162 A1 WO 2019070162A1
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somatotropin
plasmid dna
cells
growth hormone
escherichia coli
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Илья Владимирович ДУХОВЛИНОВ
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Илья Владимирович ДУХОВЛИНОВ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

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  • the invention relates to molecular biology, biotechnology, medicine and can be used for the production of human growth hormone.
  • Technical field
  • somatotropin Human growth hormone
  • somatropin somatotropic hormone
  • somatotropic hormone Human growth hormone
  • Somatotropin has a powerful anabolic and anti-catabolic effect, enhances protein synthesis and inhibits its breakdown, and also helps reduce the deposition of subcutaneous fat, increase fat burning and increase the ratio of muscle to fat.
  • somatotropin is involved in the regulation of carbohydrate metabolism - it causes a pronounced increase in blood glucose and is one of the antagonists of insulin on the effect on carbohydrate metabolism. Also described is its effect on pancreatic islet cells, an immunostimulating effect, increased calcium absorption by bone tissue, etc.
  • somatotropin improves memory and cognitive function, especially in patients with somatotropic function of the pituitary gland, and that administration of somatotropin can improve the mood and well-being of patients with low levels of somatotropin in the blood.
  • the target protein obtained is secreted or accumulated in the periplasmic space, however, a higher protein yield can be obtained if the protein accumulates in inclusion bodies.
  • Strains with this type of somatotropin accumulation are part of the above.
  • the quality of the obtained inclusion bodies depends on the yield of protein suitable for use, somatotropin monomers, upon further purification.
  • the task, which is aimed at creating the present invention is the creation of a strain-producer of somatotropin, which allows to obtain a greater yield of the monomeric form of somatotropin, compared with known strains.
  • the authors of the present invention identified using a strain based on E cells . coli BL21 (DE3) and the pET-21 (+) vector, the protein is synthesized without His-tag, in inclusion bodies, that a specific change in the T7 promoter and operator lac makes it possible to achieve a significant increase in the yield of the monomeric form of somatotropin, by 34%, from 46% up to 80%.
  • the technical result from the use of the created plasmid DNA and strain is to significantly increase the yield of the monomeric form of somatotropin. This technical effect is achieved by changing the kinetics of accumulation of the target protein in the cell due to the introduction of specific mutations in the T7 promoter and lac operator of plasmid DNA.
  • the proposed plasmid DNA pET-21a (+) somat for the synthesis of growth hormone human in Escherichia cells coli represented by pET-21a (+) vector, sequence with T7 promoter on lac operator characterized by SEQ ID NO: 3, containing the insert of the gene encoding the human growth hormone protein, characterized by the amino acid sequence SEQ ID NO: 1, optimized by the codon composition for expression in Escherichia cells coli .
  • the sequence from the T7 promoter according to the lac operator of the vector pET-21a (+), characterized by SEQ ID NO: 3, is modified: the A6G mutation is introduced into the T7 promoter, the lac operator contains the T22G, T23G mutations.
  • the sequence of the elements in the plasmid DNA is clear to the average person skilled in the art.
  • Codon composition for the organism of the expression of the target gene can be carried out manually, or using specialized software, for example, on the website molbiol.ru, or encorbio.com/protocols/Codon.htm, based on the amino acid sequence of the protein.
  • strain Escherichia coli BL21 (DE3) / pET-21a (+) - producing growth hormone human based on Escherichia cells coli BL21 (DE3), transformed with the described plasmid DNA.
  • the invention is illustrated by the following graphic materials.
  • PET-21a somat plasmid DNA electrophoregram, 0.8% agarose gel: 1 — DNA ladder GenRuler 1 kb molecular weight marker; 2 - negative control - this plasmid DNA without restriction enzyme treatment; 3 - 8 - this plasmid DNA treated with restrictases: 3 - HindIII + BglII; 4 - HindIII + NdeI; 5 - HindIII + XhoI; 6 –BglII + NdeI; 7 - BglII + XhoI; 8 - NdeI + XhoI.
  • the amino acid sequence of SEQ ID NO: 1 characterizing human growth hormone, without a signal sequence, was translated into a nucleotide sequence with simultaneous codon optimization for expression in E. coli cells using the program on the site molbiol.ru and the addition of a start and stop codon in one from variants of two stop codons, as well as restriction sites flanking the resulting gene.
  • the calculated nucleotide sequence was synthesized chemically using the ASM-800 DNA synthesizer (BIOSET, Russia).
  • the plasmid DNA fragment pET-21a (+) was also synthesized, limited to the restriction sites Bgl II and Xba I, containing the T7 promoter and lac operator, namely, its three variants, containing, respectively, the nucleotide sequences SEQ ID NO: 2 - SEQ ID NO : 4, characterizing variants of the plasmid DNA fragment pET-21a (+) with the T7 promoter by the lac operator, - (1) without mutations, (2) with the A6G mutation in the T7 promoter and with T22G mutations, T23G in the lac operator, (3) with the C7G mutation in the T7 promoter and the T16C mutation in the lac operator.
  • the obtained DNA fragments, as well as the pET-21a (+) vector were treated with appropriate restrictases according to the instructions for these enzymes.
  • the obtained fragments were purified using preparative electrophoresis and used in the ligation reaction.
  • a ligation reaction of the purified restricted DNA fragments into the purified restricted vector was carried out sequentially.
  • the reaction mixture contained 2 ⁇ l of the purified restricted DNA fragment, 3.5 ⁇ l of 10X ligase buffer (1X buffer contains 10 mM Tris-HCl (pH 7.5 at 37 ° C), 10 mM MgCl2, 50 mM NaCl, 0.1 mg / ml bovine serum albumin (BSA)) (Thermo Fisher Scientific Inc.), 3.5 ⁇ l of 50% polyethylene glycol 4000, 1 ⁇ l of a restricted vector, 5 ⁇ l of T4 ligase (Thermo Fisher Scientific Inc.).
  • the reaction mixture was brought to 35 ⁇ l with non-nucleus water and incubated at 22 ° C for 4 hours. The enzymatic reaction was stopped by incubating the reaction mixture at 65 ° C for 15 minutes.
  • the mixture was purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 ⁇ m (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.
  • Competent Escherichia coli cells of strain DH10B / R (Gibko BRL, USA) with the F-mcrA ⁇ genotype (mrr-hsdRMS-mcrBC) were prepared ⁇ 80dlacZ ⁇ M 15 ⁇ lacX74 deoR recA1 endA1 araD139 ⁇ (ara, I am I, I-I, I, I am I, I, I, I, I, I). carried out their transformation of the resulting ligase mixture.
  • E. coli cells were prepared for transformation of the obtained plasmid DNA — competent cells were obtained as follows. The cells were incubated at + 37 ° C for 16 hours in 5 ml of L-broth containing 1% tryptone, 1% yeast extract and 1% sodium chloride. The culture was diluted with fresh L-broth 50-100 times and grown on a rocking chair at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 minutes.
  • Transformation of the obtained competent cells was performed by electroporation.
  • An Eporator electroporator (Eppendorf, Germany) and sterile electroporation cuvettes (Eppendorf, Germany) were used, with a volume of 100 ⁇ l, a 1 mm slit.
  • the cells were placed in 1 ml of S ⁇ C-medium (2% bactotrypton; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2, 10 mM MgSO4; 20 mM glucose) and incubated for 40 minutes at 37 ° C, then smeared into LB-agar with antibiotic on a Petri dish and incubated for 16 h at 37 ° C.
  • S ⁇ C-medium 2% bactotrypton; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2, 10 mM MgSO4; 20 mM glucose
  • E. coli clones grown on a Petri dish with ampicillin were analyzed for the presence of plasmid DNA used in the present experiment, with simultaneous transfer of the analyzed E. coli clones to individual Petri dishes with selective medium. Plasmid DNA was isolated from such clones and analyzed by sequencing using specific primers. This allowed the selection of clones-producers developed plasmid DNA (variants). These strains were used to obtain plasmid DNA variants pET-21a (+) somat.
  • the accumulation and isolation of plasmid DNA was carried out as follows. A single colony of E. coli plasmid producer cells grown on LB agar in a petri dish with ampicillin added was inoculated into standard liquid LB (Gibko BRL, USA), 2-10 ml, containing ampicillin at a concentration of 50 ⁇ g / ml, and carried out fermentation at 37 ° C in a thermostatically controlled rotary shaker for 16 hours at 250 rpm. The obtained culture of bacterial cells was precipitated by centrifugation at 4000g for 10 minutes. at + 4 ° C.
  • plasmid DNA isolation kit (Cytokine, Russia), according to the instructions for the kit.
  • the selected plasmid DNA was analyzed by electrophoresis in a 0.8% agarose gel.
  • Example 2 The creation of a producer strain of somatotropin predominantly monomeric form
  • the mutated rne (rne131) gene encodes a truncated form of RNase E, which reduces the intracellular destruction of mRNA, leading to an increase in its enzymatic stability.
  • the lon and opm mutations in protease genes allow the production of large amounts of non-proteolyzed recombinant proteins.
  • Competent E. coli BL21 (DE3) cells were obtained and transformed with the obtained plasmid DNA (variants) as described in Example 1, instead of a ten-fold diluted ligase mixture, a solution of plasmid in water was used. The grown clones were tested for the presence of the target plasmid DNA using sequencing of the isolated plasmid DNA. As a result, a clone of E. coli cells was selected, which was further used to obtain human growth hormone (strain-producer), variants.
  • strain-producer human growth hormone
  • standard agarized LB-medium containing ampicillin at a concentration of 50 ⁇ g / ml and glucose at a concentration of 1% to block non-specific expression.
  • Induction of expression was performed when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm.
  • PYP-5052 medium was used, consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na2HPO4, 50 mM K2HPO4, 25 mM (NH4) 2SO4, 2 mM MgSO4, 0.5% glycerol, 0.05% glucose and 0.2% lactose, 0.2% lactose was used as an inducer (Studier FW. Protein Expr Pure. 2005 May; 41 (1): 207 -34)
  • PYP-5052 containing ampicillin at a concentration of 50 ⁇ g / ml, was inoculated with a single colony of the producer strain (variant). Fermentation was carried out at + 37 ° C in a thermostatic rotary type shaker at 250 vol. min within 14 hours before the absence of a significant change in OP600 for 1 hour. An aliquot was taken for analysis of the expression of the gene encoding somatotropin using PAGE, every hour, after the induction of biomass was completed, it was precipitated by centrifugation at 9000 g.
  • Induction of gene expression using IPTG was performed as follows.
  • the cells of a single producer strain colony (variants) were incubated at + 37 ° C in a thermostatic rotary type shaker at 250 rpm. for 16 h in LB medium (1% tryptone, 1% yeast extract and 1% sodium chloride) containing ampicillin at a concentration of 50 ⁇ g / ml.
  • the culture was diluted with fresh LB medium 50 times and grown in a thermostated rotary type shaker at 250 rpm. + 37 ° C until the cell culture reaches an optical density of 0.6-0.8 optical units at a wavelength of 600 nm.
  • induction of expression of the recombinant gene was performed by adding IPTG to the culture to a final concentration of 1 mM to 2 mM. Induction was carried out for 14 hours, a sample was taken for analysis using SDS-PAGE every hour, at the end of the induction, the biomass was precipitated by centrifugation.
  • the cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonylfluoride, based on 1 g of cells, 5-7 ml of buffer.
  • the cell suspension was treated with ultrasound 7 times for 30 seconds with an interval of 30 seconds (ultrasound frequency is 22 kHz), a sample was taken for SDS-PAGE analysis (PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate).
  • SDS-PAGE analysis PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate.
  • the percentage of the content of the target protein of the total protein when using the analyzed three strains producing somatotropin is shown in the graphs of Figure 1.
  • the lysate was centrifuged for 10 minutes. at + 4 ° ⁇ , 5000 g.
  • a supernatant (supernatant) and sediment sample was taken for analysis of protein localization and evaluation of its solubility using SDS-PAGE. The analysis demonstrated the insolubility of the obtained somatotropin.
  • the precipitated biomass was lysed using 3 sonication cycles for 30 seconds with a break of 2 minutes on ice. Then the inclusion bodies were washed three times with 0.2 M sodium deoxycholate, which made it possible to obtain the drug without impurities of bacterial endotoxins.
  • Inclusion bodies were dissolved in 8 M urea solution, then protein refolding was performed in refolding buffer (0.1M Tris pH 8.0, 0.2 mM EDTA) with 0.5 M L-arginine.
  • Fractions of 1 ml were collected, analyzed by electrophoresis in 20% PAAG-DDS-Na, the fractions with the target protein were combined, the protein concentration in them was determined by the Bradford method.
  • the column was calibrated using 10 mg bovine serum albumin, ovalbumin and soybean trypsin inhibitor.

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Abstract

The invention relates to molecular biology, biotechnology and medicine, and may be used to produce human growth hormone. Proposed is a plasmid DNA for the synthesis of human growth hormone in Escherichia coli cells, which is represented by the vector pET-21a(+), wherein the sequence from T7 promoter to lac operator is modified and described by SEQ ID NO:3, with a gene insert that codes for the protein described by amino acid sequence SEQ ID NO:1 and that has an optimised codon composition for expression in Escherichia coli cells. Also proposed is a human growth hormone production strain based on Escherichia coli cells BL21 (DE3) transformed with the plasmid DNA described. Using the proposed inventions makes it possible to achieve a significant increase in the output of the monomeric form of somatotropin, up to 80%.

Description

Штамм-продуцент соматотропина преимущественно мономерной формыStrain producing somatotropin mainly monomeric form
Изобретение относится к молекулярной биологии, биотехнологии, медицине и может быть использовано для производства гормона роста человека Техническая областьThe invention relates to molecular biology, biotechnology, medicine and can be used for the production of human growth hormone. Technical field
Гормон роста человека (соматотропин, соматропин, соматотропный гормон) – один из гормонов передней доли гипофиза. Вызывает выраженное ускорение линейного (в длину) роста, в основном за счет роста длинных трубчатых костей конечностей. Соматотропин оказывает мощное анаболическое и анти-катаболическое действие, усиливает синтез белка и тормозит его распад, а также способствует снижению отложения подкожного жира, усилению сгорания жира и увеличению соотношения мышечной массы к жировой. Кроме того, соматотропин принимает участие в регуляции углеводного обмена – он вызывает выраженное повышение уровня глюкозы в крови и является одним из антагонистов инсулина по действию на углеводный обмен. Описано также его действие на островковые клетки поджелудочной железы, иммуностимулирующий эффект, усиление поглощения кальция костной тканью и др. Human growth hormone (somatotropin, somatropin, somatotropic hormone) is one of the hormones of the anterior pituitary gland. Causes a pronounced acceleration of linear (in length) growth, mainly due to the growth of long tubular bones of the limbs. Somatotropin has a powerful anabolic and anti-catabolic effect, enhances protein synthesis and inhibits its breakdown, and also helps reduce the deposition of subcutaneous fat, increase fat burning and increase the ratio of muscle to fat. In addition, somatotropin is involved in the regulation of carbohydrate metabolism - it causes a pronounced increase in blood glucose and is one of the antagonists of insulin on the effect on carbohydrate metabolism. Also described is its effect on pancreatic islet cells, an immunostimulating effect, increased calcium absorption by bone tissue, etc.
Терапевтическое применение соматотропина:Therapeutic use of growth hormone:
1. Для лечения нарушений роста у детей.1. For the treatment of growth disorders in children.
2. Для лечения нервных расстройств. В некоторых работах показано, что соматотропин улучшает память и познавательные функции, особенно у больных с недостаточностью соматотропной функции гипофиза, и что введение соматотропина может улучшать настроение и самочувствие больных с низким уровнем соматотропина в крови.2. For the treatment of nervous disorders. Some studies have shown that somatotropin improves memory and cognitive function, especially in patients with somatotropic function of the pituitary gland, and that administration of somatotropin can improve the mood and well-being of patients with low levels of somatotropin in the blood.
3. Для профилактики старческих заболеваний3. For the prevention of senile diseases
4. Применение в спортивной медицине в качестве анаболического препарата.4. Application in sports medicine as an anabolic drug.
Для получения соматотропина известно использование многих штаммов Еscherichia coli – продуцентов на основе клеток W3110 (RU2287574C2, RU2433185C2, RU2337968C2, CN103173440 (A), AU731758 (B2), US5496713 (A), WO2005067601 (A2), US5932439 (A), EP0587427 (A1), CN104561020 (A)), K-12 (294, X1776, BE1201, RV308, MKD3207) (RU2337968C2, US4859600 (A), RU2287574C2, RU2433185C2, US4874703 (A), EP0089666 (A2), RU2031121C1), JM (83, 101, 105, 109) (EA200601793A1, WO2005067601 (A2), JPH0771495 (B2), AU731758 (B2)), B (RU2433185C2, EP0089666 (A2)), MT (012, 10675-10853) (AU731758 (B2), US5496713 (A), JPH09216832 (A)), HB101 (JPS60234584 (A), EP0587427 (A1), US4518690 (A)), MC1061 (JPH0771495 (B2), US6010875 (A)), ATCC №39384, 39386 (EP0173215 (A2)), D1210 (EP0067540 (A2)), HM10011-HM10020 (KR20020080108 (A)), YK537 (CN103882015 (B)), chil776 (WO2005067601 (A2)), JA221 (WO2005067601 (A2)), C41 (DE3), C43 (De3) (CN103882015 (B)), K802 (RU1248280), Rosetta, Rosetta (DE3) (CN103882015 (B)), BL21, BL21 (DE3) (RU2002133932A, WO2014046484 (A1), CN103882015 (B), WO2011103325 (A1), NZ555206 (A), WO2004005335 (A2), US2010041153 (A1), US2011237509 (A1), WO2009057622 (A1).To obtain somatotropin, it is known to use many strains of Escherichia coli — producers based on W3110 cells (RU2287574C2, RU2433185C2, RU2337968C2, CN103173440 (A), AU731758 (B2), US5496713 (A), WO2005067601 (A2), 5859- 372 (B2), 58593132 (B2), B225, 374402 ), CN104561020 (A)), K-12 (294, X1776, BE1201, RV308, MKD3207) (RU2337968C2, US4859600 (A), RU2287574C2, RU2433185C2, US4874703 (A), EP0089666 (A2), 312031202, US4874703 (A), EP 31895C2, US4874703 (A), EP 318975C2, US4874600 (A), EP2287574C2 , 101, 105, 109) (EA200601793A1, WO2005067601 (A2), JPH0771495 (B2), AU731758 (B2)), B (RU2433185C2, EP0089666 (A2)), MT (012, 10675-10853) (AU731758 (B2), US5496713 (A), JPH09216832 (A)), HB101 (JPS60234584 (A), EP0587427 (A1), US4518690 (A)), MC1061 (JPH0771495 (B2), US6010875 (A)), ATCC No. 39384, 39386 (EP0173215 ( A2)), D1210 (EP0067540 (A2)), HM10011-HM10020 (KR20020080108 (A)), YK537 (CN103882015 (B)), chil776 (WO2005067601 (A2)), JA221 (WO2005067601 (A2)), C41 (A2) , C43 (De3) (C N103882015 (B)), K802 (RU1248280), Rosetta, Rosetta (DE3) (CN103882015 (B)), BL21, BL21 (DE3) (RU2002133932A, WO2014046484 (A1), CN103882015 (B), WO2011103325 (A1), NZ, A1Z, A1, ZZ, A1, ZZ) A), WO2004005335 (A2), US2010041153 (A1), US2011237509 (A1), WO2009057622 (A1).
В ряде таких штаммов получаемый целевой белок – секретируемый, либо накапливаемый в периплазматическом пространстве, однако больший выход белка возможно получить, если белок накапливается в тельцах включения. Штаммы с таким типом накопления соматотропина составляют часть приведенных выше. Однако от качества получаемых телец включения зависит выход белка, пригодного для применения, - мономеров соматотропина, - при дальнейшей очистке. Задачей, на решение которой направлено создание настоящего изобретения, является создание штамма-продуцента соматотропина, позволяющего получить больший выход мономерной формы соматотропина, по сравнению с известными штаммами.In a number of such strains, the target protein obtained is secreted or accumulated in the periplasmic space, however, a higher protein yield can be obtained if the protein accumulates in inclusion bodies. Strains with this type of somatotropin accumulation are part of the above. However, the quality of the obtained inclusion bodies depends on the yield of protein suitable for use, somatotropin monomers, upon further purification. The task, which is aimed at creating the present invention, is the creation of a strain-producer of somatotropin, which allows to obtain a greater yield of the monomeric form of somatotropin, compared with known strains.
Известен штамм-продуцент соматотропина на основе клеток E . coli BL21 (DE3) и вектора рЕТ22b(+), белок синтезируется без His-тага, в тельцах включения (RU2002133932A). Данный штамм, а также содержащаяся в нем плазмида являются прототипом.Known producing strains of somatotropin based on E cells . coli BL21 (DE3) and the pET22b (+) vector, the protein is synthesized without His-tag, in inclusion bodies (RU2002133932A). This strain, as well as the plasmid contained in it, are the prototype.
Авторами настоящего изобретения выявлено с использованием штамма на основе клеток E . coli BL21 (DE3) и вектора рЕТ-21(+), белок синтезируется без His-тага, в тельцах включения, что специфичное изменение Т7 промотора и lac оператора позволяет достичь значительного увеличения выхода мономерной формы соматотропина, - на 34%, с 46% до 80%.The authors of the present invention identified using a strain based on E cells . coli BL21 (DE3) and the pET-21 (+) vector, the protein is synthesized without His-tag, in inclusion bodies, that a specific change in the T7 promoter and operator lac makes it possible to achieve a significant increase in the yield of the monomeric form of somatotropin, by 34%, from 46% up to 80%.
Технический результат от использования созданных плазмидной ДНК и штамма – в значительном увеличении выхода мономерной формы соматотропина. Данный технический эффект достигается благодаря изменению кинетики накопления целевого белка в клетке за счет внесения специфичных мутаций в Т7 промотор и lac оператор плазмидной ДНК.The technical result from the use of the created plasmid DNA and strain is to significantly increase the yield of the monomeric form of somatotropin. This technical effect is achieved by changing the kinetics of accumulation of the target protein in the cell due to the introduction of specific mutations in the T7 promoter and lac operator of plasmid DNA.
Технический результат от использования группы изобретений выражается также в расширении спектра плазмид и штаммов-продуцентов для получения соматотропина, что немаловажно, учитывая широкое применение данного белка. Клинический опыт показал, что, оптимизируя лечение низкорослости, целесообразно иметь в арсенале несколько аналогичных фармацевтических препаратов, получаемых различными технологиями или даже методами. Длительное лечение (годами) одним препаратом соматотропина вызывает в организме уменьшение чувствительности к нему (http://xn--80aqkgbchgfn.xn--p1ai/poluchenie-gormona-rosta-v-biotehno).. The technical result from the use of a group of inventions is also expressed in expanding the range of plasmids and producer strains for producing somatotropin, which is important, given the widespread use of this protein. Clinical experience has shown that, while optimizing the treatment of short stature, it is advisable to have in the arsenal several similar pharmaceutical preparations obtained by various technologies or even methods. Long-term treatment (for years) with a single somatotropin drug causes a decrease in sensitivity in the body (http: //xn--80aqkgbchgfn.xn--p1ai/poluchenie-gormona-rosta-v-biotehno) ..
Предложена плазмидная ДНК pET-21a(+)somat для синтеза гормона роста человека в клетках Escherichia coli, представленная вектором pET-21a(+), последовательность с Т7 промотора по lac оператор охарактеризована SEQ ID NO:3, содержащим вставку гена, кодирующего белок гормон роста человека, охарактеризованный аминокислотной последовательностью SEQ ID NO:1, оптимизированного по кодонному составу для экспрессии в клетках Escherichia coli. Последовательность с Т7 промотора по lac оператор вектора pET-21a(+), охарактеризованная SEQ ID NO:3, является модифицированной: в T7 промотор введена мутация A6G, в lac оператор – мутации T22G, T23G. Последовательность расположения элементов в плазмидной ДНК понятна среднему специалисту в данной области.The proposed plasmid DNA pET-21a (+) somat for the synthesis of growth hormone human in Escherichia cells coli , represented by pET-21a (+) vector, sequence with T7 promoter on lac operator characterized by SEQ ID NO: 3, containing the insert of the gene encoding the human growth hormone protein, characterized by the amino acid sequence SEQ ID NO: 1, optimized by the codon composition for expression in Escherichia cells coli . The sequence from the T7 promoter according to the lac operator of the vector pET-21a (+), characterized by SEQ ID NO: 3, is modified: the A6G mutation is introduced into the T7 promoter, the lac operator contains the T22G, T23G mutations. The sequence of the elements in the plasmid DNA is clear to the average person skilled in the art.
Оптимизация по кодонному составу для организма экспрессии целевого гена может быть осуществлена вручную, либо с использованием специализированного программного обеспечения, например, на сайте molbiol.ru, либо encorbio.com/protocols/Codon.htm, на основе аминокислотной последовательности белка.Optimization of the codon composition for the organism of the expression of the target gene can be carried out manually, or using specialized software, for example, on the website molbiol.ru, or encorbio.com/protocols/Codon.htm, based on the amino acid sequence of the protein.
Также предложен штамм Escherichia coli BL21 (DE3)/pET-21a(+) - продуцент гормона роста человека, на основе клеток Escherichia coli BL21 (DE3), трансформированных описанной плазмидной ДНК. Also proposed strain Escherichia coli BL21 (DE3) / pET-21a (+) - producing growth hormone human, based on Escherichia cells coli BL21 (DE3), transformed with the described plasmid DNA.
Изобретение проиллюстрировано следующими графическими материалами. The invention is illustrated by the following graphic materials.
Фиг.1. Графики, демонстрирующие кинетику накопления соматотропина при индукции экспрессии кодирующего его гена в клетках штамма-продуцента с вариантами строения Т7 промотора и lac оператора: ось абсцисс – время, часы, ось ординат – процент целевого белка от общего; график, построенный по «точкам», - кинетика накопления соматотропина при строении Т7 промотора и lac оператора как охарактеризовано SEQ ID NO:4, построенный по «треугольникам», - как охарактеризовано SEQ ID NO:2, построенный по «квадратам», - как охарактеризовано SEQ ID NO:3.1. Graphs showing the kinetics of somatotropin accumulation during the induction of expression of a gene encoding it in cells of a producer strain with variants of the T7 promoter structure and operator lac: abscissa axis — time, hours, ordinate axis — percentage of the target protein from the total; plotted by “points” —the kinetics of somatotropin accumulation in the T7 promoter and lac operator structure as described by SEQ ID NO: 4, constructed by “triangles” —as characterized by SEQ ID NO: 2, constructed by “squares” —as characterized by SEQ ID NO: 3.
Фиг.2. Электрофореграмма плазмидной ДНК pET-21a(+)somat, 0,8% агарозный гель: 1 – маркер молекулярного веса DNA ladder GeneRuler 1 kb; 2 – отрицательный контроль – данная плазмидная ДНК без обработки рестриктазами; 3-6 - данная плазмидная ДНК, обработанная рестриктазой: 3 - HindIII; 4 – BglII; 5 – Ndel; 6 – XhoI;2. PET-21a (+) somat plasmid DNA electrophoregram, 0.8% agarose gel: 1 - DNA ladder GeneRuler 1 kb molecular weight marker; 2 - negative control - this plasmid DNA without restriction enzyme treatment; 3-6 - this plasmid DNA treated with a restriction enzyme: 3 - HindIII; 4 - BglII; 5 - Ndel; 6 - XhoI;
Фиг.3. Электрофореграмма плазмидной ДНК pET-21a(+)somat, 0,8% агарозный гель: 1 – маркер молекулярного веса DNA ladder GenRuler 1 kb; 2 – отрицательный контроль – данная плазмидная ДНК без обработки рестриктазами; 3 – 8 - данная плазмидная ДНК, обработанная рестриктазами: 3 - HindIII+BglII; 4 – HindIII+NdeI; 5 – HindIII+XhoI; 6 –BglII+NdeI; 7 – BglII+XhoI; 8 – NdeI+XhoI.3. PET-21a (+) somat plasmid DNA electrophoregram, 0.8% agarose gel: 1 — DNA ladder GenRuler 1 kb molecular weight marker; 2 - negative control - this plasmid DNA without restriction enzyme treatment; 3 - 8 - this plasmid DNA treated with restrictases: 3 - HindIII + BglII; 4 - HindIII + NdeI; 5 - HindIII + XhoI; 6 –BglII + NdeI; 7 - BglII + XhoI; 8 - NdeI + XhoI.
ПримерыExamples
Авторами настоящего изобретения проведены лабораторные исследования, подтверждающие возможность реализации охарактеризованных изобретений. Полученные результаты исследований проиллюстрированы следующими примерами.The authors of the present invention conducted laboratory studies confirming the possibility of the implementation of the characterized inventions. The results of the research are illustrated by the following examples.
Пример 1. Создание плазмидной ДНК pET-21a(+)somat – вариантов с различиями в Т7 промоторе и lac оператореExample 1. Creating plasmid DNA pET-21a (+) somat - variants with differences in T7 promoter and lac operator
1.1. Создание нуклеотидной последовательности, кодирующей гормон роста человека, и вариантов фрагмента плазмидной ДНК pET-21a(+) с Т7 промотора по lac оператор1.1. The creation of a nucleotide sequence that encodes a human growth hormone, and variants of the plasmid DNA fragment pET-21a (+) from the T7 promoter according to the lac operator
Аминокислотную последовательность SEQ ID NO:1, характеризующую гормон роста человека, без сигнальной последовательности, переводили в нуклеотидную с одновременной кодонной оптимизацией для экспрессии в клетках E.coli с использованием программы на сайте molbiol.ru и добавлением старт- и стоп-кодона, в одном из вариантов двух стоп-кодонов, а также сайтов рестрикции, фланкирующих получаемый ген. Рассчитанную нуклеотидную последовательность синтезировали химическим методом с помощью синтезатора ДНК ASM-800 (БИОССЕТ, Россия).The amino acid sequence of SEQ ID NO: 1 characterizing human growth hormone, without a signal sequence, was translated into a nucleotide sequence with simultaneous codon optimization for expression in E. coli cells using the program on the site molbiol.ru and the addition of a start and stop codon in one from variants of two stop codons, as well as restriction sites flanking the resulting gene. The calculated nucleotide sequence was synthesized chemically using the ASM-800 DNA synthesizer (BIOSET, Russia).
Был синтезирован также фрагмент плазмидной ДНК pET-21a(+), ограниченный сайтами рестрикции Bgl II и Xba I, содержащий Т7 промотор и lac оператор, а именно три его варианта, содержащие, соответственно, нуклеотидные последовательности SEQ ID NO:2 - SEQ ID NO:4, характеризующие варианты фрагмента плазмидной ДНК pET-21a(+) с Т7 промотора по lac оператор, - (1) без мутаций, (2) с мутацией A6G в T7 промоторе и с мутациями T22G, T23G в lac операторе, (3) с мутацией C7G в T7 промоторе и с мутацией T16C в lac операторе.The plasmid DNA fragment pET-21a (+) was also synthesized, limited to the restriction sites Bgl II and Xba I, containing the T7 promoter and lac operator, namely, its three variants, containing, respectively, the nucleotide sequences SEQ ID NO: 2 - SEQ ID NO : 4, characterizing variants of the plasmid DNA fragment pET-21a (+) with the T7 promoter by the lac operator, - (1) without mutations, (2) with the A6G mutation in the T7 promoter and with T22G mutations, T23G in the lac operator, (3) with the C7G mutation in the T7 promoter and the T16C mutation in the lac operator.
1.2. Создание вариантов плазмидной ДНК pET-21a(+)somat1.2. Creation of variants of plasmid DNA pET-21a (+) somat
Полученные фрагменты ДНК, а также вектор pET-21a(+) (Novagen, США) обрабатывали соответствующими рестриктазами по инструкции к данным ферментам. Далее полученные фрагменты очищались с использованием препаративного электрофореза и использовались в реакции лигирования. The obtained DNA fragments, as well as the pET-21a (+) vector (Novagen, USA) were treated with appropriate restrictases according to the instructions for these enzymes. Next, the obtained fragments were purified using preparative electrophoresis and used in the ligation reaction.
Осуществлялась реакция лигирования очищенных рестрицированных фрагментов ДНК в очищенный рестрицированный вектор последовательно. Реакционная смесь содержала 2 мкл очищенного рестрицированного фрагмента ДНК, 3,5 мкл 10Х буфера для лигазы (1Х буфер содержит 10 мМ Tris-HCl (pH 7,5 при 37°C), 10 мМ MgCl2, 50 мМ NaCl, 0,1 мг/мл бычий сывороточный альбумин (БСА)) (Thermo Fisher Scientific Inc.), 3,5 мкл 50% полиэтиленгликоля 4000, 1 мкл рестрицированного вектора, 5 мкл Т4 лигазы (Thermo Fisher Scientific Inc.). Реакционная смесь доводилась до 35 мкл безнуклеазной водой и инкубировалась при 22°С в течение 4 часов. Ферментативная реакция останавливалась инкубацией реакционной смеси при 65°С в течение 15 минут. A ligation reaction of the purified restricted DNA fragments into the purified restricted vector was carried out sequentially. The reaction mixture contained 2 μl of the purified restricted DNA fragment, 3.5 μl of 10X ligase buffer (1X buffer contains 10 mM Tris-HCl (pH 7.5 at 37 ° C), 10 mM MgCl2, 50 mM NaCl, 0.1 mg / ml bovine serum albumin (BSA)) (Thermo Fisher Scientific Inc.), 3.5 μl of 50% polyethylene glycol 4000, 1 μl of a restricted vector, 5 μl of T4 ligase (Thermo Fisher Scientific Inc.). The reaction mixture was brought to 35 μl with non-nucleus water and incubated at 22 ° C for 4 hours. The enzymatic reaction was stopped by incubating the reaction mixture at 65 ° C for 15 minutes.
Смесь очищали от солей диализом на нитроцелюлозных фильтрах с диаметром пор 0,025 мкм (Millipore, США). Диализ проводили против раствора, содержащего 0,5 мМ ЭДТА в 10% глицерине, в течение 10 мин..The mixture was purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 μm (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.
1.3. Создание штамма (вариантов) на основе клеток Escherichia coli DH10B/R для амплификации полученной плазмидной ДНК (вариантов) 1.3. Creating a strain (variants) based on Escherichia coli DH10B / R cells for amplifying the obtained plasmid DNA (variants)
Подготавливали компетентные клетки Escherichia coli штамма DH10B/R (Gibko BRL, США) с генотипом F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM 15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara,leu)769 galU galKλ- rpsL nupG, и далее осуществляли их трансформацию полученной лигазной смесью. Competent Escherichia coli cells of strain DH10B / R (Gibko BRL, USA) with the F-mcrA Δ genotype (mrr-hsdRMS-mcrBC) were prepared φ80dlacZΔM 15 ΔlacX74 deoR recA1 endA1 araD139 Δ (ara, I am I, I-I, I, I am I, I, I, I, I, I, I). carried out their transformation of the resulting ligase mixture.
Подготавливали клетки Е. coli для трансформации полученной плазмидной ДНК – получали компетентные клетки - следующим образом. Инкубировали клетки при +37oС в течение 16ч в 5 мл L-бульона, содержащего 1% триптон, 1% дрожжевой экстракт и 1% натрий хлористый. Разводили культуру свежим L-бульоном в 50-100 раз и выращивали на качалке при +37oС до оптической плотности 0,2-0,3 при длине волны 590 нм. При достижении оптической плотности более 0,3 культуру разводили свежим L-бульоном до оптической плотности 0,1 и растили 30 мин. Переносили 100 мл культуры в стерильную центрифужную пробирку и осаждали клетки при +4oС на 5000g в течение 10 мин. Супернатант сливали, клетки ресуспендировали в 50 мл 0,1 М CaCl2, охлажденного на льду. Инкубировали клетки 20 мин на льду. Осаждали клетки в течение 10 мин. при 5000 об./мин, +4oС. Супернатант сливали, клетки ресуспендировали в 3 мл 0,1 М СаСl2, охлажденного на льду. E. coli cells were prepared for transformation of the obtained plasmid DNA — competent cells were obtained as follows. The cells were incubated at + 37 ° C for 16 hours in 5 ml of L-broth containing 1% tryptone, 1% yeast extract and 1% sodium chloride. The culture was diluted with fresh L-broth 50-100 times and grown on a rocking chair at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 minutes. Transferred 100 ml of the culture to a sterile centrifuge tube and precipitate the cells at + 4 ° C for 5000 g for 10 minutes. The supernatant was discarded, the cells were resuspended in 50 ml of 0.1 M CaCl2, cooled on ice. Incubated cells for 20 min on ice. The cells were precipitated for 10 min. at 5000 rpm, + 4 ° C. The supernatant was discarded, the cells were resuspended in 3 ml of 0.1 M CaCl2, cooled on ice.
Трансформацию полученных компетентных клеток осуществляли методом электропорации. Использовали электропоратор Eporator (Eppendorf, Германия) и стерильные кюветы для электропорации (Eppendorf, Германия), объемом 100 мкл, щель 1 мм. Transformation of the obtained competent cells was performed by electroporation. An Eporator electroporator (Eppendorf, Germany) and sterile electroporation cuvettes (Eppendorf, Germany) were used, with a volume of 100 μl, a 1 mm slit.
К 12 мкл компетентных клеток добавляли 1 мкл десятикратно разведенной лигазной смеси, осуществляли перемешивание и перенос в кювету, которую помещали в электропоратор. Трансформацию проводили при электрическом импульсе напряженностью 1,7 кВ длительностью 5 мсек. После трансформации клетки помещали в 1 мл SОС-cреды (2% бактотриптон; 0,5% дрожжевой экстракт; 10 мМ NaCl; 2,5 мМ KCl; 10 мМ MgCl2, 10 мМ MgSO4; 20 мМ глюкоза) и инкубировали в течение 40 минут при 37°С, после чего вмазывали в LB-агар с антибиотиком на чашке Петри и инкубировали 16 ч при 37°С.1 μl of a ten-fold diluted ligase mixture was added to 12 μl of competent cells, mixed and transferred to a cuvette, which was placed in an electroporator. The transformation was carried out with an electric pulse with a strength of 1.7 kV and a duration of 5 ms. After transformation, the cells were placed in 1 ml of SОC-medium (2% bactotrypton; 0.5% yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2, 10 mM MgSO4; 20 mM glucose) and incubated for 40 minutes at 37 ° C, then smeared into LB-agar with antibiotic on a Petri dish and incubated for 16 h at 37 ° C.
Выросшие на чашке Петри с ампициллином клоны E.coli анализировали на наличие используемой в настоящем эксперименте плазмидной ДНК, с одновременным переносом анализируемых клонов клеток E.coli на отдельные чашки Петри с селективной средой. Из таких клонов выделяли плазмидную ДНК и анализировали секвенированием с использованием специфичных праймеров. Это позволило отобрать клоны-продуценты разработанных плазмидных ДНК (вариантов). Данные штаммы использовали для получения вариантов плазмидной ДНК pET-21a(+)somat. E. coli clones grown on a Petri dish with ampicillin were analyzed for the presence of plasmid DNA used in the present experiment, with simultaneous transfer of the analyzed E. coli clones to individual Petri dishes with selective medium. Plasmid DNA was isolated from such clones and analyzed by sequencing using specific primers. This allowed the selection of clones-producers developed plasmid DNA (variants). These strains were used to obtain plasmid DNA variants pET-21a (+) somat.
1.4. Получение вариантов плазмидной ДНК pET-21a(+)somat1.4. Obtaining variants of plasmid DNA pET-21a (+) somat
Наработку и выделение плазмидной ДНК осуществляли следующим образом. Единичную колонию клеток продуцента плазмид E.coli, выращенную на LB-агаре в чашке Петри с добавлением ампициллина, инокулировали в стандартную жидкую среду LB (Gibko BRL, США), 2–10 мл, содержащую ампициллин в концентрации 50 мкг/мл, и осуществляли ферментацию при 37°С в термостатированном шейкере роторного типа в течение 16 ч при 250 об./мин. Полученную культуру бактериальных клеток осаждали центрифугированием при 4000g в течение 10 мин. при +4°С. Удаляли супернатант и из осадка клеток выделяли плазмидную ДНК с помощью набора для выделения плазмидной ДНК (Цитокин, Россия), по инструкции к набору. Выделенную плазмидную ДНК анализировали электрофорезом в 0,8%-ном агарозном геле.The accumulation and isolation of plasmid DNA was carried out as follows. A single colony of E. coli plasmid producer cells grown on LB agar in a petri dish with ampicillin added was inoculated into standard liquid LB (Gibko BRL, USA), 2-10 ml, containing ampicillin at a concentration of 50 μg / ml, and carried out fermentation at 37 ° C in a thermostatically controlled rotary shaker for 16 hours at 250 rpm. The obtained culture of bacterial cells was precipitated by centrifugation at 4000g for 10 minutes. at + 4 ° C. The supernatant was removed and plasmid DNA was isolated from the cell pellet using plasmid DNA isolation kit (Cytokine, Russia), according to the instructions for the kit. The selected plasmid DNA was analyzed by electrophoresis in a 0.8% agarose gel.
Пример 2. Создание штамма-продуцента соматотропина преимущественно мономерной формыExample 2. The creation of a producer strain of somatotropin predominantly monomeric form
2.1. Создание штамма-продуцента гормона роста человека на основе клеток Escherichia coli BL21 (DE3) (вариантов)2.1. Creation of a producer strain of human growth hormone based on Escherichia coli BL21 (DE3) cells (variants)
Выделенными согласно описанной в Примере 1 методике вариантами плазмидной ДНК pET-21a(+)somat, с различиями в строении T7 промотора и lac оператора трансформировали клетки E.coli штамма BL21 (DE3) (Invitrogen, USA), с генотипом F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3), содержащие в геноме λDe3 лизоген и мутацию rne131. Мутированный ген rne (rne131) кодирует усеченную форму РНКазы Е, что уменьшает внутриклеточное разрушение мРНК, приводя к увеличению ее ферментативной стабильности. lon- и оmpТ-мутации по генам протеаз позволяют получать непротеолизированные рекомбинантные белки в больших количествах. The variants of plasmid DNA pET-21a (+) somat, identified in Example 1, with differences in the structure of the T7 promoter and operator lac transformed E. coli cells of the strain BL21 (DE3) (Invitrogen, USA), with the genotype F- ompT hsdSB ( rB-mB-) gal dcm rne131 (DE3), containing λDe3 in the genome lysogen and mutation rne131. The mutated rne (rne131) gene encodes a truncated form of RNase E, which reduces the intracellular destruction of mRNA, leading to an increase in its enzymatic stability. The lon and opm mutations in protease genes allow the production of large amounts of non-proteolyzed recombinant proteins.
Получали компетентные клетки Е.coli BL21(DE3) и трансформировали их полученной плазмидной ДНК (вариантами) как описано в Примере 1, вместо десятикратно разведенной лигазной смеси использовали раствор плазмиды в воде. Выросшие клоны проверяли на наличие целевой плазмидной ДНК с использованием секвенирования выделенной плазмидной ДНК. В итоге выбрали клон клеток Е.coli, который далее использовали для получения гормона роста человека (штамм-продуцент), варианты.Competent E. coli BL21 (DE3) cells were obtained and transformed with the obtained plasmid DNA (variants) as described in Example 1, instead of a ten-fold diluted ligase mixture, a solution of plasmid in water was used. The grown clones were tested for the presence of the target plasmid DNA using sequencing of the isolated plasmid DNA. As a result, a clone of E. coli cells was selected, which was further used to obtain human growth hormone (strain-producer), variants.
2.2. Выявление штамма-продуцента соматотропина преимущественно мономерной формы из созданных вариантов2.2. Detection of somatotropin producer strain of predominantly monomeric form from the created variants
2.2.1. Индукция синтеза соматотропина в штамме-продуценте (вариантах)2.2.1. Induction of somatotropin synthesis in the producer strain (variants)
Индукцию экспрессии получали и с использованием ИПТГ, и 0,2% лактозы. Induction of expression was obtained using IPTG, and 0.2% lactose.
2.2.1.1. при индукции синтеза соматотропина 0.2% лактозой по методу Штудиера2.2.1.1. in the induction of somatotropin synthesis with 0.2% lactose by the method of Studier
Для культивирования полученных штаммов-продуцентов использовали стандартную агаризованную LB-среду, содержащую ампициллин в концентрации 50 мкг/мл и глюкозу в концентрации 1% для блокирования неспецифической экспрессии. For the cultivation of the obtained producer strains, standard agarized LB-medium was used containing ampicillin at a concentration of 50 μg / ml and glucose at a concentration of 1% to block non-specific expression.
Индукцию экспрессии проводили при достижении культурой клеток оптической плотности 0.6-0.8 оптических единиц при длине волны 600 нм. Induction of expression was performed when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm.
Для автоиндукции экспрессии по методу Штудиера использовали среду PYP-5052, состоящую из 1% пептона (Gibco, США), 0.5% дрожжевого экстракта (Gibco, США), 50 мМ Na2HPO4, 50 мМ K2HPO4, 25 мМ (NH4)2SO4, 2 мМ MgSO4, 0.5% глицерола, 0.05% глюкозы и 0.2% лактозы, в качестве индуктора использовали 0.2% лактозу (Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005 May;41(1):207-34).For autoinduction of expression by the method of Studier, PYP-5052 medium was used, consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na2HPO4, 50 mM K2HPO4, 25 mM (NH4) 2SO4, 2 mM MgSO4, 0.5% glycerol, 0.05% glucose and 0.2% lactose, 0.2% lactose was used as an inducer (Studier FW. Protein Expr Pure. 2005 May; 41 (1): 207 -34)
В среду PYP-5052, содержащую ампициллин в концентрации 50 мкг/мл, инокулировали единичную колонию штамма-продуцента (варианта). Ферментацию проводили при +37°С в термостатированном шейкере роторного типа при 250 об. мин. в течение 14 часов до отсутствия существенного изменения ОП600 за 1 час. Отбирали аликвоту на анализ экспрессии гена, кодирующего соматотропин, методом электрофореза в ПААГ, каждый час, по окончании индукции биомассу осаждали центрифугированием при 9000g.On Wednesday, PYP-5052, containing ampicillin at a concentration of 50 μg / ml, was inoculated with a single colony of the producer strain (variant). Fermentation was carried out at + 37 ° C in a thermostatic rotary type shaker at 250 vol. min within 14 hours before the absence of a significant change in OP600 for 1 hour. An aliquot was taken for analysis of the expression of the gene encoding somatotropin using PAGE, every hour, after the induction of biomass was completed, it was precipitated by centrifugation at 9000 g.
2.2.1.2. при индукции синтеза соматотропина ИПТГ2.2.1.2. with induction of somatotropin IPTG synthesis
Индукцию экспрессии гена с использованием ИПТГ осуществляли следующим образом. Инкубировали клетки единичной колонии штамма-продуцента (вариантов) при +37°С в термостатированном шейкере роторного типа при 250 об./мин. в течение 16ч в LB среде (1% триптон, 1% дрожжевой экстракт и 1% натрий хлористый), содержащей ампициллин в концентрации 50 мкг/мл. Разводили культуру свежей LB средой в 50 раз и выращивали в термостатированном шейкере роторного типа при 250 об./мин. +37°С до достижения культурой клеток оптической плотности 0.6-0.8 оптических единиц при длине волны 600 нм. После этого осуществляли индукцию экспрессии рекомбинантного гена добавлением ИПТГ к культуре до конечной концентрации от 1 мМ до 2 мМ. Индукцию проводили в течение 14 часов, отбирали пробу для анализа с использованием SDS-PAGE каждый час, по окончании индукции биомассу осаждали центрифугированием.Induction of gene expression using IPTG was performed as follows. The cells of a single producer strain colony (variants) were incubated at + 37 ° C in a thermostatic rotary type shaker at 250 rpm. for 16 h in LB medium (1% tryptone, 1% yeast extract and 1% sodium chloride) containing ampicillin at a concentration of 50 μg / ml. The culture was diluted with fresh LB medium 50 times and grown in a thermostated rotary type shaker at 250 rpm. + 37 ° C until the cell culture reaches an optical density of 0.6-0.8 optical units at a wavelength of 600 nm. After this, induction of expression of the recombinant gene was performed by adding IPTG to the culture to a final concentration of 1 mM to 2 mM. Induction was carried out for 14 hours, a sample was taken for analysis using SDS-PAGE every hour, at the end of the induction, the biomass was precipitated by centrifugation.
2.2.2. Анализ отобранных проб2.2.2. Sampling Analysis
Клетки ресуспендировали в лизирующем буфере, содержащем 20 мМ трис-НСl рН 7,5, 5 мМ ЭДТА и 1 мМ феноксиметилсульфонилфторид, из расчета на 1 г клеток 5-7 мл буфера. Суспензию клеток обрабатывали ультразвуком 7 раз по 30 сек с интервалом в 30 сек (частота ультразвука составляет 22 кГц), отбирали пробу на анализ SDS-PAGE (PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate). Процент содержания целевого белка от общего белка при использовании анализируемых трех штаммов-продуцентов соматотропина приведен на графиках Фиг.1. The cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonylfluoride, based on 1 g of cells, 5-7 ml of buffer. The cell suspension was treated with ultrasound 7 times for 30 seconds with an interval of 30 seconds (ultrasound frequency is 22 kHz), a sample was taken for SDS-PAGE analysis (PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate). The percentage of the content of the target protein of the total protein when using the analyzed three strains producing somatotropin is shown in the graphs of Figure 1.
Лизат центрифугировали 10 мин. при +4°С, 5000 g. Отбирали пробу надосадочной жидкости (супернатанта) и осадка для анализа локализации белка и оценки его растворимости, с использованием SDS-PAGE. Анализ продемонстрировал нерастворимость полученного соматотропина. The lysate was centrifuged for 10 minutes. at + 4 ° С, 5000 g. A supernatant (supernatant) and sediment sample was taken for analysis of protein localization and evaluation of its solubility using SDS-PAGE. The analysis demonstrated the insolubility of the obtained somatotropin.
2.2.1.3. Описание кинетики накопления соматотропина2.2.1.3. Description of growth hormone kinetics
Кинетика накопления соматотропина в клетках штамма-продуцента (вариантов) была выявлена с использованием дензитометрического сканирования электрофореграмм спектра белков, полученного из лизатов клеток E.coli штамма-продуцента (вариантов) после индукции экспрессии, с помощью программы ImageJ, и оказалась сходной при индукции экспрессии 0,2% лактозой и ИПТГ (от 1 мМ до 2 мМ) и приведена на Фиг.1.The kinetics of somatotropin accumulation in producer strain cells (variants) was detected using densitometric scanning of protein spectrum electrophorems obtained from lysates of E. coli producer strain (variants) after induction of expression using the ImageJ program and was found to be similar in the expression induction 0 , 2% lactose and IPTG (from 1 mm to 2 mm) and is shown in Figure 1.
2.2.2. Рефолдинг2.2.2. Refolding
После окончания индукции осажденную биомассу лизировали с помощью 3 циклов соникации по 30 сек с перерывом в 2 мин на льду. Затем трехкратно отмывали тельца включения 0.2 М дезоксихолятом натрия, что позволяло получить препарат без примесей бактериальных эндотоксинов.After the end of induction, the precipitated biomass was lysed using 3 sonication cycles for 30 seconds with a break of 2 minutes on ice. Then the inclusion bodies were washed three times with 0.2 M sodium deoxycholate, which made it possible to obtain the drug without impurities of bacterial endotoxins.
Растворяли тельца включения в 8 М растворе мочевины, затем осуществляли рефолдинг белка в буфере для рефолдинга (0.1M Tris pH 8.0, 0.2 mM ЭДТА) с 0.5 М L-аргинином. Inclusion bodies were dissolved in 8 M urea solution, then protein refolding was performed in refolding buffer (0.1M Tris pH 8.0, 0.2 mM EDTA) with 0.5 M L-arginine.
Проводили хроматографическую очистку полученного раствора белка. Около 1200 мл обессоленного супернатанта помещали на 20 мл S-Sepharose колонку, уравновешенную 20 мМ Tris-HCl pH 8.0. Колонку отмывали 200 мл 50 мМ Tris pH 8.0, белок элюировали 160 мл линейного градиента 50 мМ Tris pH 8.0 1М NaCl. Фракция очищенного на S-Sepharose гормона роста человека была помещена на S-100 колонку (2.5*80 см), предварительно уравновешенную фосфатным буфером. Собирали фракции по 1 мл, анализировали электрофоретически в 20% ПААГ-ДДс-Na, фракции с целевым белком объединяли, концентрацию белка в них определяли по методу Бредфорд. Колонку откалибровывали с использованием 10 мг бычьего сывороточного альбумина, овальбумина и соевого трипсинового ингибитора. Conducted chromatographic purification of the resulting protein solution. About 1200 ml of the desalted supernatant were placed on a 20 ml S-Sepharose column equilibrated with a 20 mM Tris-HCl pH 8.0. The column was washed with 200 ml of 50 mM Tris pH 8.0, the protein was eluted with 160 ml of a linear gradient of 50 mM Tris pH 8.0 with 1 M NaCl. The fraction of purified human S-Sepharose growth hormone was placed on an S-100 column (2.5 * 80 cm), previously equilibrated with phosphate buffer. Fractions of 1 ml were collected, analyzed by electrophoresis in 20% PAAG-DDS-Na, the fractions with the target protein were combined, the protein concentration in them was determined by the Bradford method. The column was calibrated using 10 mg bovine serum albumin, ovalbumin and soybean trypsin inhibitor.
Формы соматотропина анализировали с использованием электрофореза в ПААГ при нанесении препаратов рефолдированного соматотропина, полученных с использованием трех штаммов и двух типов индукторов.Forms of somatotropin were analyzed using electrophoresis in PAAG when applying preparations of refold somatotropin, obtained using three strains and two types of inductors.
Удалось получить наибольший процент мономерной формы соматотропина, 80%, при использовании штамма-продуцента со строением Т7 промотора и lac оператора как охарактеризовано SEQ ID NO:3. 46% Мономерной формы соматотропина получили с использованием штамма-продуцента со строением Т7 промотора и lac оператора как охарактеризовано SEQ ID NO:2, т.е. оригинального для вектора pET-21a(+). При внесении же мутаций в данные элементы как охарактеризовано SEQ ID NO:4 получили значительное уменьшение выхода мономерной формы соматотропина при рефолдинге, до 21%. Полученные данные подтверждают нетривиальность предложенного решения. It was possible to obtain the highest percentage of monomeric form of somatotropin, 80%, using a producer strain with the structure of the T7 promoter and operator lac as characterized by SEQ ID NO: 3. 46% of the monomeric form of somatotropin was obtained using a producer strain with the structure of the T7 promoter and the operator lac as characterized by SEQ ID NO: 2, i.e. original for the vector pET-21a (+). When making the same mutations in these elements as described by SEQ ID NO: 4, a significant decrease in the yield of the monomeric form of somatotropin during refolding was obtained, up to 21%. The data obtained confirm the non-triviality of the proposed solution.
Таким образом, оптимальным для получения высокого процента мономерной формы соматотропина является использование штамма Escherichia coli BL21 (DE3)/pET-21a(+), со строением Т7 промотора и lac оператора плазмидной ДНК pET-21a(+) как охарактеризовано SEQ ID NO:3. Thus, it is optimal to obtain a high percentage of monomeric form of somatotropin using the Escherichia coli BL21 (DE3) / pET-21a (+) strain, with the structure of the T7 promoter and the pAC-21a (+) plasmid DNA operator as characterized by SEQ ID NO: 3 .
Пример 3. Характеристика штамма-продуцента соматотропина преимущественно мономерной формыExample 3. Characterization of a somatotropin producing strain of predominantly monomeric form
3.1. Рестрикционный анализ3.1. Restriction analysis
Из полученного штамма-продуцента мономерной формы соматотропина выделяли плазмидную ДНК как описано выше.From the obtained producer strain of the monomeric form of somatotropin, plasmid DNA was isolated as described above.
При обработке выделенной плазмидной ДНК эндонуклеазами рестрикции наблюдали картину, приведенную на Фиг. 2 и 3, которая характеризует созданный штамм-продуцент соматотропина преимущественно мономерной формы. Соответствие полученных результатов ожидаемым было подтверждено.During the processing of the isolated plasmid DNA by restriction endonucleases, the pattern shown in FIG. 2 and 3, which characterizes the created somatotropin producing strain of predominantly monomeric form. The correspondence of the obtained results to the expected was confirmed.
3.2. Культуральные характеристики3.2. Cultural characteristics
Культуральные свойства Грам-отрицательные прямые палочки, размером 1,1-1,5-2,0-3,0 мкм, одиночные, спор и капсул не образуют. Каталазоположительные. Оксидазоотрицательные. Факультативные анаэробы. Интервал pH 5-7. Катализируют D-глюкозу и некоторые другие углеводы с образованием кислоты и газа, не сбраживают лактозу, арабинозу и галактозу. Реакция Фогес-Проскауэра отрицательная, не образуют H2S, но гидролизуют мочевину.Cultural properties Gram-negative straight sticks, 1.1-1.5-2.0-3.0 microns in size, single, do not form spores and capsules. Catalase positive. Oxy-negative. Facultative anaerobes. The pH range is 5-7. They catalyze D-glucose and some other carbohydrates with the formation of acid and gas, do not ferment lactose, arabinose and galactose. The reaction Voges-Proskauer negative, do not form H2S, but hydrolyze urea.
Ростовые характеристики Клетки растут в интервале температур от 8°С до 43°С, интервал для культивирования – 28-38°С, оптимум роста при 37°С.Growth characteristics Cells grow in the temperature range from 8 ° C to 43 ° C, the interval for cultivation is 28-38 ° C, the optimum growth at 37 ° C.
Описание визуальных и цитологических наблюдений при стандартных условиях культивирования Клетки хорошо растут на простых питательных средах, содержащих и не содержащих ампициллин. На агаризованной среде – колонии гладкие, круглые, слабо выпуклые, с ровным краем. В жидких средах образуют равномерную светорассеивающую суспензию, при хранении без перемешивания оседают на дно. Description of visual and cytological observations under standard culture conditions. Cells grow well on simple nutrient media containing and not containing ampicillin. On agar medium - colonies are smooth, round, slightly convex, with an even edge. In liquid media, they form a uniform light-scattering suspension; during storage they are deposited to the bottom without stirring.

Claims (2)

  1. Плазмидная ДНК pET-21a(+)somat для синтеза гормона роста человека в клетках Escherichia coli, представленная вектором pET-21a(+), последовательность с Т7 промотора по lac оператор охарактеризована SEQ ID NO:3, содержащим вставку гена, кодирующего белок гормон роста человека, охарактеризованный аминокислотной последовательностью SEQ ID NO:1, оптимизированного по кодонному составу для экспрессии в клетках Escherichia coli. Plasmid DNA pET-21a (+) somat for the synthesis of growth hormone sequence in Escherichia coli cells , represented by pET-21a (+), sequence from T7 promoter by lac operator characterized by SEQ ID NO: 3, containing the insert of the gene encoding the human growth hormone protein, characterized by the amino acid sequence SEQ ID NO: 1, optimized by codon composition for expression in Escherichia coli cells.
  2. Штамм Escherichia coli BL21 (DE3)/pET-21a(+) - продуцент гормона роста человека на основе клеток Escherichia coli BL21 (DE3), трансформированных плазмидной ДНК по п.1.The strain of Escherichia coli BL21 (DE3) / pET-21a (+) - producer of growth hormone human based on Escherichia coli BL21 (DE3) cells transformed with plasmid DNA according to claim 1.
PCT/RU2018/050040 2017-10-08 2018-04-12 Production strain of somatotropin, primarily in monomeric form WO2019070162A1 (en)

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RU2233879C1 (en) * 2002-12-17 2004-08-10 Институт биоорганической химии им. академиков М.М.Шемякина и Ю.А.Овчинникова РАН Recombinant plasmid dna pes1-6 encoding polypeptide somatotropin and strain escherichia coli bl21(de3)/pes1-6 as producer of recombinant somatotropin
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