WO2022052368A1 - 一种新甘精胰岛素原及其制备甘精胰岛素的方法 - Google Patents

一种新甘精胰岛素原及其制备甘精胰岛素的方法 Download PDF

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WO2022052368A1
WO2022052368A1 PCT/CN2020/138950 CN2020138950W WO2022052368A1 WO 2022052368 A1 WO2022052368 A1 WO 2022052368A1 CN 2020138950 W CN2020138950 W CN 2020138950W WO 2022052368 A1 WO2022052368 A1 WO 2022052368A1
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glargine
proinsulin
insulin glargine
insulin
peptide
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French (fr)
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汤传根
王璟
潘尚书
范晓阳
刘晓锐
陈松
张昊宁
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Amphastar Nanjing Pharma Co Ltd
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Amphastar Nanjing Pharma Co Ltd
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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
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6408—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a new proinsulin glargine and a method for preparing insulin glargine, belonging to the technical field of recombinant protein preparation.
  • Insulin is a hormone that regulates glucose metabolism in animals. This hormone is composed of two peptide chains, A chain and B chain. The A chain has 21 amino acids and the B chain has 30 amino acids, for a total of 51 amino acids. Among them, A 7 (Cys)-B 7 (Cys), A 20 (Cys)-B 19 (Cys) 4 cysteines form 2 disulfide bonds, connecting the A chain and the B chain. In the A chain there is an intrachain disulfide bond formed by A 6 (Cys) and A 11 (Cys). Diabetes is characterized by elevated blood sugar levels due to a lack of insulin, the only hormone in the body that lowers blood sugar, and/or increased hepatic glucose production.
  • the overall goal of insulin analog development is to mimic physiological insulin secretion, thereby improving glycemic control in patients with type 1 and type 2 diabetes (Berger MA comment. Diabetes Res Clin Pract. 6:S25–S31, 1989; Sanlioglu AD et al., Clinical utility of insulin and insulin analogs. Islets 5(2):67-78, 2013).
  • Recent insulin analogs include the addition or replacement of amino acid residues on the natural insulin molecule by genetic engineering or biochemical reactions, or modifications to other functional groups. These modifications alter the rate of bioavailability by altering the pharmacological, pharmacokinetic, and pharmacodynamic properties of insulin molecules, such as insulin aspart, glargine, lispro, and others.
  • Insulin glargine (US Pat. No. 5,656,722) is a long-acting insulin analog.
  • the substitution of glycine for aspartic acid at A 21 and the addition of two arginine residues to the C-terminus of the B-chain is to allow insulin glargine to form a precipitate (hexamer-microcrystal) upon injection.
  • the isoelectric point of insulin glargine increases from pH 5.4 to pH 6.7, making the molecule water soluble at acidic pH, eventually leading to the slow dissociation of insulin glargine hexamers into monomers.
  • insulin glargine In the neutral pH subcutaneous region, hexamer formation results in slow, non-peak dissolution and absorption of insulin from the injection site, providing a long duration of action that lasts 24-26 hours.
  • the prolonged action of insulin glargine reduces the peak effect and reduces the risk of hypoglycemia.
  • insulin glargine Compared with NPH neutral protamine zinc insulin, insulin glargine showed a lower incidence of severe hypoglycemia (Sanlioglu AD et al., Clinical utility of insulin and insulin analogs. Islets 5(2):67-78, 2013 ).
  • Human insulin was the first protein-based drug produced by recombinant DNA technology. In 1978, human insulin was first successfully expressed in the laboratory; in 1982, recombinant human insulin was approved as a therapeutic drug.
  • the precursor protein of recombinant human insulin is synthesized by genetically modified biosynthesis and cleaved by proteolysis to generate active insulin. Almost all insulin analogs on the market are genetically engineered from the human insulin gene and produced in E. coli or yeast.
  • Patent CN103981242A relates to a method for producing insulin using copper/zinc superoxide dismutase SOD as a fusion peptide.
  • the fusion peptide consists of 64 amino acids, of which the first amino acid is Met and the last amino acid is Arg; meanwhile, all cysteine residues in the fusion peptide chain are replaced by serine residues.
  • the amino acid sequence of the SOD fusion peptide fragment in this patent is as follows: MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPR (SEQ ID NO: 1). There are 5 Lys residues in this SOD fragment, which is the natural cleavage site of trypsin. In the subsequent enzymatic cleavage process, miscut impurities are easily generated, resulting in lower yield and purity.
  • U.S. Patent No. 6,777,207 relates to a proinsulin peptide structure, which consists of a shortened C-peptide (not more than 15 amino acid residues in length), which has two molecules as glycine-arginine or glycine-lysine. a terminal amino acid, which is attached to the A-carboxy-terminal chain.
  • the proinsulin construct contains a 29 amino acid B chain in contrast to the full length 30 amino acid B chain of native human insulin.
  • the invention discloses a new proinsulin glargine capable of effectively improving the preparation process of recombinant insulin glargine and a method for preparing the insulin glargine.
  • the structure of proinsulin glargine designed by the present invention includes an N-terminal fusion peptide sequence (SOD after site-directed mutagenesis), an insulin A chain modified with A 21 and a full-length human insulin B chain containing two arginine residues.
  • SOD after site-directed mutagenesis N-terminal fusion peptide sequence
  • an insulin A chain modified with A 21 and a full-length human insulin B chain containing two arginine residues.
  • the A21 amino acid of the A chain of insulin glargine is replaced with glycine for asparagine, and two arginine residues are added to the carboxy terminus of the B chain.
  • the structure of the proinsulin glargine adopts an "OC peptide" strategy, ie there is no C peptide sequence between the B and A chains.
  • the proinsulin glargine is expressed in Escherichia coli, and then renatured together with the fusion peptide under correct renaturation conditions, and then the fusion peptide and the insulin glargine molecule are separated by trypsin digestion, thereby obtaining glargine insulin.
  • the proinsulin glargine designed by the invention can be effectively folded into its natural structure in the presence of the SOD fragment fusion peptide after site-directed mutation, and the fermentation yield of insulin glargine can be improved.
  • a suitable protease cleavage site for proinsulin increasing the C-peptide sequence may lead to residues of C-peptide after cleavage, and affect the purification and yield.
  • the newly invented proinsulin glargine adopts the "OC peptide" strategy, which can effectively avoid the residue of C peptide after enzymatic cleavage, and minimize the mass loss in the enzymatic cleavage conversion step.
  • the first object of the present invention is to provide a new proinsulin glargine, the amino acid sequence of which has the following structure:
  • RR 1 is a fusion peptide sequence, and the amino acid sequence of R is: MATX 1 AVSVLKGDGPVQGIINFEQX 2 ESNGPVKVWGSIX 3 GLTEGLHGFHVHEFGDNTAGSTSAGP;
  • X 1 is proline Pro(P) or histidine His(H);
  • X 2 is proline Pro(P) or histidine His(H);
  • X 3 is proline Pro(P) or histidine Amino acid His(H);
  • R 1 is arginine Arg(R) or lysine Lys(K);
  • B 1 -B 32 are two arginine Arg residues (R) added after the C-terminus at position B 30 of the B chain B 1 -B 30 of native human insulin;
  • a 1 -A 20 are insulin A chains with 20 amino acids
  • a 21 is glycine ( G ).
  • amino acid sequence of R is as shown in MATPAVSVLKGDGPVQGIINFEQPESNGPVKVWGSIPGLTEGLHGFHVHEFGDNTAGSTSAGP (SEQ ID NO:2); or MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP (SEQ ID NO:3).
  • the C-terminus of the fusion peptide sequence is linked to B1 - B32 by a lysine residue or an arginine residue.
  • amino acid sequence of A1 - A20 is GIVEQCCTSICSLYQLENYC (SEQ ID NO:4).
  • the amino acid sequence of B1 - B30 is FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 5).
  • the B chain is extended with two arginine residues such that the amino acid sequence of B1-B32 is FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR .
  • amino acid sequence of (B 1 -B 32 )-(A 1 -A 20 )-A 21 in proinsulin glargine is:
  • the amino acid sequence of proinsulin glargine RR 1 -(B 1 -B 32 )-(A 1 -A 20 )-A 21 is SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID Any of NO:9 and SEQ ID NO:10:
  • the second object of the present invention is to provide DNA encoding proinsulin glargine.
  • the DNA has the nucleotide sequence shown in any one of SEQ ID NOs: 11-14.
  • the SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 encode SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, respectively: 9 and the amino acid sequence shown in SEQ ID NO:10.
  • a third aspect of the present invention provides an expression vector containing the DNA.
  • the expression vector includes, but is not limited to, the pET series of plasmids.
  • a fourth object of the present invention is to provide non-plant cells, including but not limited to eukaryotic cells or prokaryotic cells, expressing said proinsulin glargine.
  • the cell expresses the DNA encoding proinsulin glargine, or incorporates the expression vector.
  • the microbial cells include, but are not limited to, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cells.
  • the cells comprise mammalian cells or insect cells.
  • the cells are prokaryotic cells, including but not limited to Escherichia coli, Bacillus subtilis, or any improved varieties more suitable for recombinant protein expression, such as Escherichia coli DH5a, K12JM107, W3110, BL21(DE3), Rosetta or other strains.
  • the cell is a recombinant E. coli containing the pET28a plasmid carrying the gene encoding proinsulin glargine.
  • the fourth aspect of the present invention is to provide a method for producing insulin glargine, which utilizes the recombinant Escherichia coli to ferment and produce insulin glargine.
  • the method is to inoculate recombinant E. coli in BFM medium and ferment at 35-37° C. for at least 20 hours.
  • the BFM medium contains diammonium hydrogen phosphate, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, citric acid monohydrate, glucose, yeast powder and trace elements.
  • the inoculation is inoculation with a recombinant E. coli seed solution.
  • the seed liquid undergoes two-stage fermentation; the first-stage fermentation is in LB medium, fermented at 35-37° C. for 6-10 h to obtain the first-grade seed liquid; and then the first-stage seed liquid is fermented at 0.2 % of the inoculum was transferred to BFM medium, and cultured for 6-10 h to obtain secondary seed liquid.
  • the method further cleaves, renatures and purifies the fermented insulin glargine.
  • the method includes the steps of:
  • step (3) (4) using trypsin to digest the modified proinsulin glargine in step (3), and obtain insulin glargine by acid hydrolysis;
  • the step (2) adopts lysozyme treatment and high pressure homogenization.
  • the step (2) is diluted and renatured at 15-25° C. and pH 10.0-11.6.
  • the step (3) adds citraconic anhydride to the renatured proinsulin.
  • the modified proinsulin containing the sequence of claim 1 is converted by trypsin digestion, and the fusion peptide is removed, so that the lysine at position B29 is Insulin glargine modified with citraconic anhydride residues.
  • the citraconic anhydride residue is removed by acid hydrolysis at pH 1.5-2.5 to obtain insulin glargine.
  • the step (5) uses ion exchange chromatography to purify insulin glargine to obtain high-purity insulin glargine;
  • the step (5) uses preparative HPLC chromatography to further purify insulin glargine to obtain higher purity insulin glargine;
  • the high-purity insulin glargine is further crystallized and dried to form the final insulin glargine API.
  • the above-mentioned optimized gene is connected to a suitable carrier, such as pTAC expression plasmid series, pGEX series or pET series, preferably pET series plasmid, more preferably plasmid pET-28a; this plasmid can be transfected into K12 JM109 engineering bacteria or K12 W110 engineered bacteria to form expression clones.
  • a suitable carrier such as pTAC expression plasmid series, pGEX series or pET series, preferably pET series plasmid, more preferably plasmid pET-28a; this plasmid can be transfected into K12 JM109 engineering bacteria or K12 W110 engineered bacteria to form expression clones.
  • the expression plasmid is transfected into BL21(DE3) engineered bacteria.
  • the recombinant E. coli is grown to an appropriate concentration by shake flasks or fermentors, and then the expression of proinsulin glargine is induced.
  • cells containing inclusion bodies of proinsulin are lysed by lysozyme treatment and high pressure homogenization; the isolated inclusion bodies are washed with a solution containing detergent or a low concentration of chaotropic agent and treated with high pH buffer solution dissolves.
  • the pH of the lysis buffer is 11.6-12.4, the lysis buffer contains Tris, EDTA and L-cysteine; the concentration of Tris is 10-50 mM and the concentration of EDTA is 0.05-1.0 mM , the concentration of L-cysteine was 0.25-5.0 mM.
  • the concentration of Tris is 20-30 mM; the concentration of EDTA is 0.05-0.25 mM, the concentration of L-cysteine is 0.25-1.0 mM; the pH is 11.8-12.2.
  • the temperature of the solubilization buffer is 10-30°C, or 15-25°C; and the inclusion body solubilization time is 10-120 min, or 10-60 min.
  • the pH of the lysis buffer is 10.0-11.6, or 10.8-11.4; the temperature of the solution is about 10-25°C, or 15-20°C; the concentration of total protein is 1-10 g/L, or 1-7g/L; renaturation duration is 12-48h, or 24-36h.
  • a protecting reagent is used to modify proinsulin glargine before enzymatic cleavage;
  • the protecting reagent can be an electrophile that is easily reacted with the ⁇ -NH 2 group of B29-Lys, such as an acid anhydride, including but not limited to Acetic anhydride, citric anhydride or citraconic anhydride.
  • a molar excess of citraconic anhydride is added to proinsulin; alternatively, for proinsulin, a 10-fold or more molar amount of citraconic anhydride is added; or a 20-fold or more molar amount is added Multimolar amounts of citraconic anhydride.
  • the reaction temperature for modification by adding a protective reagent is 15-25° C.
  • the pH is 8.0-9.0
  • the duration is 2-8 h.
  • ethanolamine can be added to neutralize excess citraconic anhydride, the volume of ethanolamine is 40%-80% of that of citraconic anhydride, and the termination time is 10-30 min.
  • the protein concentration of the renaturation solution is adjusted to 1-7 g/L.
  • Trypsin preferably bovine trypsin
  • pH was adjusted to 9.0, and digested at 20°C for 24 hours to obtain citraconic acid residues of insulin glargine. The process was monitored by HPLC-RP (C18).
  • the cleavage reaction can be terminated by adding hydrochloric acid and adjusting the pH to 2.0-2.5.
  • the solution was kept at this low pH for 24 hours to hydrolyze the citraconic acid residues on B29-Lys to yield insulin glargine.
  • citraconic anhydride by volume to the renatured protein solution, adjust the pH to 8.5, and react at 20°C 2 hours.
  • 60% of the volume of citraconic anhydride used for the modification was added with ethanolamine for neutralization, the pH was adjusted to 9.4, and the neutralization was performed for 15 min.
  • trypsin at a final concentration of 0.063 mg/g protein was added to the citraconic acid-modified insulin glargine, pH was adjusted to 9.0, and reacted at 20°C for 24 hours to obtain glargine with citraconic acid residues insulin.
  • zinc ions with a final concentration of 1-10 mM are added, and the pH is adjusted to 5.5-6.5, so that insulin glargine is precipitated and precipitated. Purification is performed to obtain the final insulin glargine product.
  • one-step purification is performed using preparative RP-HPLC with ammonium dihydrogen phosphate buffer system, wherein the concentration of diammonium hydrogen phosphate is 0.05-0.3M, or 0.05-0.2M; pH value is 2.0-5.0 range; the organic modifier can be ethanol, methanol or acetonitrile; insulin glargine is eluted in a linear concentration gradient of organic solvent.
  • final purification is performed using preparative RP-HPLC and Tris-HCl buffer system, wherein Tris concentration is 0.02-0.3M, or 0.02-0.2M; pH value is in the range of 7.0-9.0; organic modification
  • the reagent can be ethanol, methanol or acetonitrile; insulin glargine is eluted in a linear concentration gradient of organic solvent.
  • insulin glargine eluted from RP-HPLC is precipitated using isoelectric precipitation and the precipitate is collected.
  • the collected precipitate was resuspended and washed with 0-1.5% sodium chloride solution, and the wet solid obtained by centrifugation after washing was dissolved with 40-200 mM hydrochloric acid solution, and the concentration of insulin glargine was adjusted to 20-40 mg /mL, adjust the pH to 3.0-5.0, filter the filtrate, freeze-dry it, and prepare the insulin glargine bulk drug in the form of crystals or solidified API.
  • the present invention also claims the application of the method in the preparation of insulin glargine or a medicine containing insulin glargine.
  • the invention uses the SOD fragment after site-directed mutation as the fusion peptide and adopts the "OC peptide" strategy, so that higher fermentation yield can be obtained, and the fermentation yield of insulin glargine is increased by 75% or more, and the highest is increased by 78%;
  • the present invention avoids residues of C peptide residues, reduces the quality loss in the enzymatic conversion step, reduces impurities caused by wrong refolding and wrong enzymatic cleavage in the patent application, and improves the yield of the final product and purity, the chromatographic purity of the main peak is over 99.4, and the maximum single impurity is less than or equal to 0.16%.
  • the chromatographic purity of the main peak is as high as 99.9%, and the maximum single impurity is controlled below 0.05%; the chromatographic purity of the main peak is increased by 7% relative to the comparative example, The maximum single impurity content is reduced from 2.15% to ⁇ 0.16%, which is obviously reduced by an order of magnitude.
  • the enzymatic cleavage treatment is performed by bovine trypsin, and compared with pig trypsin, the enzymatic cleavage conversion rate can be increased by 16%. Therefore, the production cost of preparing high-quality insulin glargine can be greatly reduced.
  • Fig. 1 is according to the specific example 6 of the present invention, adopts the bacterial strain purification liquid chromatogram after gene sequence SEQ ID NO:11 is constructed.
  • Fig. 2 is according to the specific example 7 of the present invention, adopts the bacterial strain purification liquid chromatogram after gene sequence SEQ ID NO:12 is constructed.
  • Fig. 3 is according to the specific example 8 of the present invention, adopts the bacterial strain purification liquid chromatogram after gene sequence SEQ ID NO:13 is constructed.
  • Figures 4A, 4B and 4C are liquid chromatographic atlases after renaturation of the strain WCB01 constructed using the gene sequence SEQ ID NO: 10 according to specific examples 1-3 of the present invention.
  • Figures 5A, 5B and 5C are liquid chromatographic atlases after renaturation of the strain WCB02 constructed using the gene sequence SEQ ID NO: 15 according to specific examples 1-3 of the present invention.
  • Fig. 6 is according to the specific example 1-3 of the present invention, adopts the liquid chromatogram after the renaturation of the bacterial strain WCB03 after gene sequence SEQ ID NO:16 is constructed.
  • Fig. 7 is according to the specific example 1-3 of the present invention, adopts the liquid chromatogram after the renaturation of the bacterial strain WCB04 after gene sequence SEQ ID NO:17 is constructed.
  • Fig. 8 is according to the specific example 1-3 of the present invention, adopts the liquid chromatogram after the renaturation of the bacterial strain WCB05 after gene sequence SEQ ID NO:18 is constructed.
  • Fig. 9A is according to the specific example 1-5 of the present invention, adopts the liquid chromatogram after the purification of strain WCB01 after gene sequence SEQ ID NO:10 is constructed.
  • Fig. 9B is according to the specific comparative example 1-3 of the present invention, adopts the liquid chromatogram after the purification of strain WCB02 after gene sequence SEQ ID NO:15 is constructed.
  • 10A is a liquid chromatographic chart of the enzymatic cleavage effect of bovine trypsin on proinsulin glargine in specific example 4 according to the present invention.
  • FIG. 10B is a liquid chromatogram of the enzymatic cleavage effect of porcine trypsin on proinsulin glargine according to the specific comparative example 4 of the present invention.
  • the sequence of the improved proinsulin glargine adopts the "OC peptide" strategy, that is, there is no amino acid sequence between the B chain and the A chain.
  • the N-terminal leader amino acid sequence enhances expression and protects proinsulin glargine from degradation by E. coli.
  • the amino acid sequence of R in fusion peptide RR1 is MATHAVSVLKGDGPVQGIINFEQHESNPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP (as shown in SEQ ID NO:3).
  • the C-terminus of the amino acid sequence of the fusion peptide is linked to the B chain of insulin glargine through an arginine or lysine residue, and finally the fusion peptide is removed by trypsin cleavage.
  • Example 2 Construction of a recombinant plasmid containing a new proinsulin encoding gene
  • the sequence of the new proinsulin was designed according to the method of Example 1: the sequence of (B 1 -B 32 )-(A 1 -A 20 )-A 21 in the new proinsulin is SEQ ID NO:6.
  • the full sequence of the fusion peptide of proinsulin glargine is SEQ ID NO: 10.
  • the genetic codon was optimized. The optimized gene sequence is shown in SEQ ID NO: 14, which contains a 5'NcoI site (CCATGG) and a 3'Hind III site (AAGCTT).
  • the DNA fragment of SEQ ID NO: 14 was chemically synthesized by a commercial CRO company, the DNA fragment was cleaved with NcoI and Hind III restriction enzymes, inserted into the pET-28a expression vector cleaved with the same restriction endonucleases, and ligated by ligase ligated to form the pET-PIG-1 expression vector.
  • the recombinant expression vector pET-PIG-1 constructed in Example 2 was transfected into E. coli BL21 (DE3) competent cells. Positive clones were screened by kanamycin resistance and confirmed by DNA sequencing. Positive clones were cultured and expanded at 37°C, and then sterile medium and glycerol were added to the cells. Transfer 1 mL of cell culture solution into sterile ampoules and store at -80°C to form a working seed bank of proinsulin glargine (WCB01).
  • the WCB01 obtained in Example 3 was inoculated into MLB medium (containing 15g/L yeast powder and 5g/L sodium chloride) at an inoculation amount of 0.2%, and cultivated for 6-14h at 37°C and 250rpm to obtain first-class seeds. liquid.
  • the first-class seed liquid was inoculated in BFM medium (containing 6g/L diammonium hydrogen phosphate, 4g/L ammonium chloride, 13.5g/L potassium dihydrogen phosphate, 1.39g/L magnesium sulfate heptahydrate, and 0.2% inoculum amount.
  • the volume ratio of 1:10 was inoculated into the BFM medium of the fermenter, and the growth temperature was 30-39°C, the growth dissolved oxygen was 10-50%, and the growth pH was 6.0-7.3 for 12-18h until the OD 600 of the fermentation broth was 100-200, add IPTG with a final concentration of 0.1-1.0mM to the fermenter to induce proinsulin glargine expression, and other growth conditions remain unchanged. Induce continuously for 8-16h, and collect the cells by a centrifuge.
  • the bacterial cells containing the inclusion bodies of proinsulin were resuspended with 25 mM Tris, 10 mM EDTA, pH 8.0 buffer, and the concentration of the bacterial cells was controlled to be 200 g/L.
  • the cells were lysed by lysozyme treatment and high-pressure homogenization, the cell lysate was centrifuged to collect the inclusion body precipitate, and the supernatant was removed.
  • Inclusion bodies were washed with 25 mM Tris, 1 M urea, 1% Tween 20, pH 8.0 washing solution. After washing, the inclusion bodies were resuspended with 25 mM Tris, 0.1 mM EDTA and 0.5 mM L-cysteine buffer, adjusted to pH 12.0, and dissolved at 15 °C for 50 min. The solubilized solution was named inclusion body solubilization solution.
  • the inclusion body dissolving solution of WCB01 prepared in Example 4 was filtered with a 1 ⁇ m PP filter element, the temperature was controlled to 20° C., the pH was adjusted to 11.0, and then renatured for 32 h to obtain renatured proinsulin glargine.
  • citraconic anhydride by volume of the renaturing solution is added for modification. Adjust the pH of the renaturation solution to 8.5, and stir to modify for 2h. After the modification was completed, ethanolamine containing 60% of the citraconic anhydride used for the modification was added to neutralize the excess citraconic anhydride, the pH was adjusted to 9.4, and the neutralization was performed for 15 min. Then, bovine trypsin with a final concentration of 0.063 mg bovine trypsin/g protein was directly added, the pH was adjusted to 9.0, and the enzyme was digested at 20° C. for 24 h. The fusion peptide was removed to obtain insulin glargine modified with citraconic anhydride.
  • Enzyme cleavage engineering was monitored by RP-HPLC (C18). When the cleavage was completed, the cleavage reaction was terminated by adjusting the pH to 2.0 with hydrochloric acid. And kept at pH 2.0 for 12h, the lysine modified by citraconic anhydride at the 29th position of B was hydrolyzed to obtain insulin glargine. After the hydrolysis was completed, zinc chloride with a final concentration of 3 mM was added, and the pH was adjusted to 6.0, so that insulin glargine formed a flocculent precipitate.
  • the precipitation of insulin glargine after enzymatic cleavage and hydrolysis in step (1) was dissolved with acetic acid with a volume fraction of 3%, and the dissolution pH value was 3.5. Dissolved insulin glargine is loaded as a sample onto a cation chromatography column and equilibrated with buffer. Insulin glargine can be eluted by a linear gradient of 30% isopropanol, 1.0 M sodium chloride. After purification by cation chromatography, the final concentration of 3mM zinc chloride was used to adjust the pH to 7.3, so that insulin glargine formed a flocculent precipitate. Repeat the above operation 2 more times.
  • the cation-chromatographic purified insulin glargine was loaded onto a reverse-phase preparative chromatography column. Equilibrate the column with a solution obtained by mixing 0.1 M ammonium dihydrogen phosphate and acetonitrile at a ratio of 9:1 and adjusting the pH to 3.5.
  • the elution buffer is a solution obtained by mixing a mixed solution of 0.1 M diammonium hydrogen phosphate and 10% acetonitrile with a pH of 3.5 and 60% acetonitrile in different proportions. Insulin glargine was eluted by a linear gradient of elution buffer. The purity of insulin glargine in the obtained insulin glargine eluate was 97%.
  • the final concentration of 3mM zinc chloride was used to adjust the pH to 7.3 to make insulin glargine form a flocculation precipitate.
  • the insulin glargine precipitate was dissolved in 3% acetic acid at a pH of 3.5.
  • Dissolved insulin glargine is loaded as a sample onto a reversed-phase preparative chromatography column. The column was equilibrated with the solution obtained by mixing 0.05M Tris and acetonitrile at a ratio of 9:1 and adjusting the pH to 8.5.
  • the elution buffer is a mixed solution of 0.05M Tris at pH 8.5 and 10% acetonitrile and 60% acetonitrile in different proportions.
  • the insulin glargine was eluted by a linear gradient of the elution buffer, and the purity of the insulin glargine in the insulin glargine eluate was determined to be 99.9%.
  • the pH was adjusted to 7.3 with 100 mM hydrochloric acid solution, and insulin glargine formed a flocculated precipitate.
  • the collected precipitate was resuspended and washed 3 times with 0.3% sodium chloride solution (pH 7.0), and centrifuged to obtain insulin glargine wet solid.
  • Refrigeration control the set temperature is -30°C, the set time is 240min, and the duration is 240min;
  • Pre-vacuum pre-vacuum 0.2000mbar, set alarm vacuum 0.5000mbar, alarm vacuum duration 10s;
  • the final insulin glargine raw material drug is obtained, and the purity is more than 99.9%.
  • the specific embodiment is the same as in Examples 2 to 6, except that the full sequence of the fusion peptide of insulin glargine is shown in SEQ ID NO: 7, and the gene sequence encoding the fusion peptide of insulin glargine is shown as SEQ ID NO: 11 shown.
  • the constructed recombinant E. coli was fermented and produced under the same conditions, and the expression level of proinsulin glargine fusion protein was 5.8 g/L.
  • the purified product was detected by HPLC, and the chromatographic purity of the high main peak of 99.42% and the maximum single impurity purity of 0.16% were obtained. Chromatographic purity (see Figure 1 and Table 1 for details).
  • Table 1 is the main peak area and retention time detected by chromatography
  • the specific embodiment is the same as Examples 2 to 6, the difference is that the full sequence of the pro-insulin glargine fusion peptide is as shown in SEQ ID NO: 8, and the gene sequence encoding the pro-insulin glargine fusion peptide is as shown in SEQ ID NO: 12 shown.
  • the constructed recombinant E. coli was fermented and produced under the same conditions, and the expression level of proinsulin glargine fusion protein was 6.1g/L.
  • the purified product was detected by HPLC and obtained 99.59% high main peak chromatographic purity and 0.14% maximum single impurity Chromatographic purity (see Figure 2 and Table 2 for details).
  • the specific embodiment is the same as in Examples 2 to 6, except that the full sequence of the fusion peptide of insulin glargine is shown in SEQ ID NO: 9, and the gene sequence encoding the fusion peptide of insulin glargine is shown as SEQ ID NO: 13 shown.
  • the constructed recombinant E. coli was fermented and produced under the same conditions, and the expression level of the proinsulin glargine fusion protein was 6.0g/L.
  • the purified product was detected by HPLC, and the chromatographic purity of the high main peak of 99.80% and the maximum single impurity purity of 0.09% were obtained. Chromatographic purity (see Figure 3 and Table 3 for details).
  • the specific embodiment is the same as Examples 2 to 3, except that the amino acid sequence in the fusion peptide RR 1 is replaced by MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPR (SEQ ID NO:1), and the DNA fragment encoding the entire amino acid sequence of SEQ ID NO:1 is adjusted accordingly:
  • nucleotide sequence encoding the above amino acid sequence was digested with plasmid pET-28a with NcoI and Hind III restriction enzymes, and the digested fragment was connected with the vector to obtain the recombinant expression vector pET-PIG-2.
  • the recombinant expression vector pET-PIG-2 was transformed into E. coli BL21(DE3) competent cells. Positive clones were screened for kanamycin resistance and confirmed by DNA sequencing. Positive clones were cultured and expanded, and then sterile medium and glycerol were added to the cells. Transfer 1 mL of cell culture solution into sterile ampoules and store at -80°C to form a working seed bank of proinsulin glargine (WCB02).
  • the recombinant strain WCB02 obtained in Comparative Example 1 was cultured in three batches according to the method described in Example 4, and processed to obtain an inclusion body lysate, which was then renatured according to the method described in Example 5. HPLC was used to detect the yields of proinsulin regenerating precursors obtained by fermenting WCB01 and WCB02, respectively. The results of three parallel experiments are shown in Table 4.
  • the renaturation liquid chromatogram of WCB01 is shown in Figures 4A, 4B and 4C, and the relevant data is shown in Tables 4A, 4B and 4C;
  • the renaturation liquid chromatogram of WCB02 is shown in Figures 5A, 5B and 5C, and the relevant data is shown in the table 5A, 5B and 5C.
  • Table 1 show that using the SOD fragment of site-directed mutagenesis as the fusion peptide and the preferred sequence SEQ ID NO: 10 using the "OC peptide" strategy, more efficient expression and more stable high fermentation yield can be obtained.
  • the fermentation yield of insulin was increased by 75% and above, with a maximum increase of 78%.
  • Table 4A Main peak area and retention time detected by chromatography
  • Table 5A Main peak area and retention time detected by chromatography
  • Table 5B Main peak areas and retention times detected by chromatography
  • Example 1 The strategy of Example 1 was adjusted to design a sequence encoding insulin glargine, and expressed in host cells, so that proinsulin glargine contained a fusion peptide R sequence that had undergone a site-directed mutation on the basis of SEQ ID NO: 15, and the amino acid sequence Designed to:
  • Refolding was performed as described in Example 5. Utilize HPLC to detect the output of the pro-insulin glargine renaturation precursor obtained by the fermentation of the strain, and the result shows that the yield of the pro-insulin glargine renaturation precursor under the same conditions is 1.9 g/L.
  • the renaturation liquid chromatograms are shown in Figure 6, respectively, and the relevant data are shown in Table 6.
  • Example 1 The strategy of Example 1 was adjusted to design a sequence encoding insulin glargine, and expressed in host cells, so that proinsulin glargine contained a fusion peptide R sequence that had undergone 2 site-directed mutagenesis on the basis of SEQ ID NO: 15, and the amino acid sequence are designed as:
  • Example 1 The strategy of Example 1 was adjusted to design a sequence encoding insulin glargine and expressed in host cells so that proinsulin glargine contained "C-peptide” (EAR), and the amino acid sequence was designed as:
  • the inclusion body lysates prepared in Comparative Example 2 were renatured according to the conditions of Example 5, respectively.
  • the renatured sample was then modified, digested and purified according to the method in Example 6.
  • the purified liquid chromatograms of the recombinant bacteria WCB01 and the recombinant bacteria WCB02 of Comparative Example 2 are shown in Figure 9A and Figure 9B, respectively, and the relevant data are shown in Tables 10A and 10B, respectively.
  • the results showed that the use of site-directed mutagenesis of 3 amino acid SOD fragments as fusion peptides and the strain (WCB01) using the "OC peptide" strategy could obtain 99.93% high main peak chromatographic purity and 0.05% maximum single heterochromatographic purity.
  • the chromatographic purities of the main peak and the largest single hetero of the strain using the unmutated SOD fragment as the fusion peptide (WCB02) were 92.25% and 2.15%, respectively.
  • the mutated SOD fragment and adopting the "0C peptide" strategy although the purity of the main peak is improved a little, the maximum single impurity content is significantly reduced by an order of magnitude, avoiding the residue of C-peptide residues, reducing the mass loss of enzyme digestion and conversion and miscut impurities .
  • Table 10A Main peak areas and retention times detected by chromatography
  • Table 10B Main peak area and retention time detected by chromatography
  • the renatured solution of WCB01 prepared in Example 3 was subjected to enzyme cleavage transformation using porcine trypsin.
  • the liquid chromatographic chromatograms of enzyme digestion and conversion using porcine trypsin and bovine trypsin are shown in Figure 10A and Figure 10B, respectively, and the relevant data are shown in Tables 11A and 11B, respectively.
  • Fig. 10A the area of the main peak with a peak time of 20.447 min is 9569627 mAU*min, and the area of the main peak with a peak appearance time of 20.730 min in Fig. 10B is 11142487 mAU*min.
  • Table 11A Main peak areas and retention times detected by chromatography
  • Table 11B Main peak areas and retention times detected by chromatography

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Abstract

本发明公开了一种新甘精胰岛素原及其制备甘精胰岛素的方法，属于重组蛋白制备技术领域。本发明设计了包含定点突变的SOD融合肽和"0C肽"的甘精胰岛素原的序列，构建了表达甘精胰岛素的重组大肠杆菌工程菌，并通过诱导工程菌表达以包涵体形式甘精胰岛素融合蛋白，再经过变性、复性、修饰、酶切和分离纯化得到成熟的甘精胰岛素原料药。本发明通过突变SOD融合肽序列将甘精胰岛素的发酵产率提高了75％；通过采用"0C肽"策略，避免C-肽残基的残留，减少酶切转化的质量损失和错切杂质。本发明制备的甘精胰岛素原料药的纯度高达99.9％，最大单杂控制在0.05％。

Description

一种新甘精胰岛素原及其制备甘精胰岛素的方法 技术领域
本发明涉及一种新甘精胰岛素原及其制备甘精胰岛素的方法，属于重组蛋白制备技术领域。
背景技术
胰岛素是一种调节动物体内葡萄糖代谢的激素。这种激素是由A链和B链2条肽链组成，A链有21个氨基酸和B链有30个氨基酸，共51个氨基酸。其中A 7(Cys)-B 7(Cys)，A 20(Cys)-B 19(Cys)4个半胱氨酸形成2个二硫键，连接A链和B链。在A链中有A 6(Cys)和A 11(Cys)形成的链内二硫键。糖尿病的特点是由于胰岛素缺乏和/或肝葡萄糖产量的增加而导致血糖水平升高，胰岛素是机体内唯一降低血糖的激素。
胰岛素类似物发展的总体目标是模拟生理性的胰岛素分泌，从而改善1型和2型糖尿病患者的血糖控制(Berger M.A comment.Diabetes Res Clin Pract.6:S25–S31,1989；Sanlioglu AD等人,Clinical utility of insulin and insulin analogs.Islets 5(2):67-78,2013)。近期的胰岛素类似物(天然胰岛素的类似物)包括由基因工程或生物化学反应在天然胰岛素分子上额外附加氨基酸残基或者替换氨基酸残基，或对其他功能组的修饰。这些修饰通过改变胰岛素分子的药理学、药代动力学和药效动力学特性，改变了生物药效率的速度，如门冬胰岛素、甘精胰岛素、赖脯胰岛素等。甘精胰岛素(美国专利US5656722)是一种长效作用的胰岛素类似物。在A 21处用甘氨酸取代门冬氨酸，在B链的C末端添加两个精氨酸残基，是为了让甘精胰岛素在注射时形成沉淀物(六聚体-微晶体)。甘精胰岛素的等电点从pH5.4提高至pH6.7，使分子在酸性pH下是水溶性的，最终导致甘精胰岛素六聚体缓慢地解离成单体。在中性pH皮下区域，六聚体的形成导致胰岛素从注射部位缓慢，无峰值溶解和吸收，提供持续24-26小时长的持续作用时间。甘精胰岛素的延长作用降低了峰值效应，降低了低血糖的风险。与NPH中性精蛋白锌胰岛素相比，甘精胰岛素显示出较低的严重低血糖发生率(Sanlioglu AD et al.,Clinical utility of insulin and insulin analogs.Islets 5(2):67-78,2013)。
人胰岛素是重组DNA技术产生的第一个蛋白类药物。1978年，人胰岛素在实验室中首次成功表达；1982年，重组人胰岛素被批准作为治疗药物。重组人胰岛素的前体蛋白由遗传修饰的生物合成，并通过蛋白酶水解切割生成活性胰岛素。几乎所有在公开销售的胰岛素类似物都是用基因工程技术从胰岛素人类基因中改良出来的，并在大肠杆菌或酵母中产生。
对于使用转基因大肠杆菌的方法，有一种方法是在大肠杆菌中分别表达胰岛素的A链和 B链，然后在体外混合磺化的A链和B链形成链间二硫键(Rich,D.H.等人，Pierce Chemical Company,Rockford.Pp.721-728,1981.和Frank,B.H.等人，Pierce Chemical Company,Rockford.Pp.729-738,1981)。然而，该方法存在缺点，因为它需要两个单独的发酵过程并形成正确的二硫键。磺化的A链和B链之间形成的不正确的二硫键会导致胰岛素的低产率。
专利CN103981242A中涉及了一种使用铜/锌超氧化物歧化酶SOD作为融合肽生产胰岛素的方法。该融合肽由64个氨基酸组成，其中第一位氨基酸为Met，最后一位氨基酸为Arg；同时，融合肽链中的半胱氨酸残基均被丝氨酸残基取代。该专利中SOD融合肽片段的氨基酸序列如下所示：MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPR(SEQ ID NO:1)。该SOD片段中有5个Lys残基，为胰蛋白酶的天然切割位点。在后续的酶切过程中，容易产生错切杂质，导致产率和纯度较低。
一种在C-肽区域中不具有保守的二元基氨基酸末端序列的胰岛素原的使用方法也被报道。例如，美国专利US6777207所涉及的胰岛素原肽结构体，它是由缩短的C-肽(长度不超过15个氨基酸残基)组成，其具有作为甘氨酸-精氨酸或甘氨酸-赖氨酸的两个末端氨基酸，其连接至A-羧基末端的链。与天然人胰岛素的全长30个氨基酸的B链相反，所述胰岛素原构建体含有29个氨基酸的B链。这种缩短的B链和缩短的C-肽在人胰岛素生产中的潜在影响尚不清楚，但是需要通过收率较低的转肽反应将B30位置的氨基酸连接上去。该专利中讲述的宿主细胞是酵母，没有证据表明该构建体可用于大肠杆菌。由于酵母表达体系的发酵周期较长会使其表达效率降低且成本升高，现在急需设计一种更高效和更完善的新型甘精胰岛素原的结构序列，以及由该结构序列制备甘精胰岛素的工艺，解决现有技术中存在的上述问题。
发明内容
本发明公开一种能够有效改善重组甘精胰岛素制备工艺的新甘精胰岛素原及其制备甘精胰岛素的方法。本发明设计的甘精胰岛素原的结构包括一个N末端融合肽序列(定点突变后的SOD)和具有A 21修饰的胰岛素A链以及含两个精氨酸残基的全长人胰岛素B链。与天然人胰岛素相比，甘精胰岛素A链的A 21位氨基酸用甘氨酸取代天冬酰胺，并且在B链的羧基末端添加两个精氨酸残基。所述甘精胰岛素原的结构采用“0C肽”策略，即B链和A链之间没有C肽序列。将该甘精胰岛素原在大肠杆菌中表达，再与融合肽一起在正确的复性条件下进行复性，再通过用胰蛋白酶酶切实现融合肽与甘精胰岛素分子的分离，从而得到甘精胰岛素。
本发明设计的甘精胰岛素原在有定点突变后的SOD片段融合肽存在的情况下，可以有效地折叠成其天然结构，提高甘精胰岛素的发酵产量。甘精胰岛素原在有合适的蛋白酶酶切位 点存在时，增加C肽序列可能会导致酶切后C肽残留，并影响纯化及收率。本发明中，新发明的甘精胰岛素原采用“0C肽”策略，可有效避免酶切后C肽的残留，并使酶切转化步骤中的质量损失最小化。
本发明的第一个目的是提供一种新的甘精胰岛素原，其氨基酸序列具有如下结构：
R-R 1-(B 1-B 32)-(A 1-A 20)-A 21
其中：
R-R 1为融合肽序列，R的氨基酸序列为：MATX 1AVSVLKGDGPVQGIINFEQX 2ESNGPVKVWGSIX 3GLTEGLHGFHVHEFGDNTAGSTSAGP；
X 1为脯氨酸Pro(P)或组氨酸His(H)；X 2为脯氨酸Pro(P)或组氨酸His(H)；X 3为脯氨酸Pro(P)或组氨酸His(H)；
R 1为精氨酸Arg(R)或赖氨酸Lys(K)；
B 1-B 32为在天然人胰岛素的B链B 1-B 30的B 30位C-末端后增加了两个精氨酸Arg残基(R)；
A 1-A 20为具有20个氨基酸的胰岛素A链；
A 21是 甘氨酸( G)。
在一种实施方式中，R的氨基酸序列如MATPAVSVLKGDGPVQGIINFEQPESNGPVKVWGSIPGLTEGLHGFHVHEFGDNTAGSTSAGP(SEQ ID NO:2)；或MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP(SEQ ID NO:3)所示。
在一种实施方式中，融合肽序列的C端通过一个赖氨酸残基或者精氨酸残基与B 1-B 32相连接。
在一种实施方式中，A 1-A 20的氨基酸序列为GIVEQCCTSICSLYQLENYC(SEQ ID NO:4)。
在一种实施方式中，B 1-B 30的氨基酸序列为FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO:5)。
在一种实施方式中，用两个精氨酸残基延长B链，使B 1-B 32的氨基酸序列为FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR。
在一种实施方式中，甘精胰岛素原中(B 1-B 32)-(A 1-A 20)-A 21的氨基酸序列为：
Figure PCTCN2020138950-appb-000001
在一种实施方式中，甘精胰岛素原R-R 1-(B 1-B 32)-(A 1-A 20)-A 21的氨基酸序列为SEQ ID NO: 7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10中的任意一种：
Figure PCTCN2020138950-appb-000002
本发明的第二个目的是提供编码甘精胰岛素原的DNA。
在一种实施方式中，所述DNA具有SEQ ID NO:11～14任一所示的核苷酸序列。
在一种实施方式中，所述SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14分别编码SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10所示的氨基酸序列。
本发明第三方面提供含有所述DNA的表达载体。
在一种实施方式中，所述表达载体包括但不限于pET系列质粒。
本发明的第四个目的是提供表达所述甘精胰岛素原的非植物细胞，包括但不限于真核细胞或原核细胞。
在一种实施方式中，所述细胞表达所述编码甘精胰岛素原的DNA，或引入了所述表达载体。
在一种实施方式中，所述微生物细胞包括但不限于大肠杆菌、枯草芽孢杆菌、酿酒酵母细胞。
在一种实施方式中，所述细胞包括哺乳动物细胞或昆虫细胞。
在一种实施方式中，所述细胞为原核细胞，包括但不限于大肠杆菌、枯草杆菌，或者是任何更适合重组蛋白表达的改良品种，如大肠杆菌DH5a、K12JM107、W3110、BL21(DE3)、Rosetta或其它菌株。
在一种实施方式中，所述细胞为重组大肠杆菌，含有携带甘精胰岛素原编码基因的pET28a质粒。
本发明的第四方面是提供一种生产甘精胰岛素的方法，利用所述重组大肠杆菌发酵生产甘精胰岛素。
在一种实施方式中，所述方法是将重组大肠杆菌接种于BFM培养基中，于35～37℃下发酵至少20h。
在一种实施方式中，所述BFM培养基含有磷酸氢二铵、氯化铵、磷酸二氢钾、七水硫酸镁、一水柠檬酸、葡萄糖、酵母粉和微量元素。
在一种实施方式中，所述接种是接种重组大肠杆菌种子液。
在一种实施方式中，所述种子液经过两级发酵；第一级发酵是在LB培养基中，于35～37℃发酵6～10h获得一级种子液；再将一级种子液以0.2％的接种量转接至BFM培养基中，培养6～10h获得二级种子液。
在一种实施方式中，所述方法还对发酵后的甘精胰岛素酶切、复性和纯化。
在一种实施方式中，所述方法包括如下步骤：
(1)将所述重组大肠杆菌培养、发酵，表达甘精胰岛素原；
(2)包涵体释放和溶解，以及甘精胰岛素原的复性；
(3)用柠康酐修饰重折叠的甘精胰岛素原；
(4)采用胰蛋白酶酶切步骤(3)修饰后的甘精胰岛素原，通过酸性水解得到甘精胰岛素；
(5)甘精胰岛素的纯化；
(6)甘精胰岛素的沉淀、洗涤、溶解、过滤和冻干。
在一种实施方式中，所述步骤(2)采用溶菌酶处理和高压均质。
在一种实施方式中，所述步骤(2)在15-25℃和pH10.0-11.6条件下稀释复性。
在一种实施方式中，所述步骤(3)向复性的甘精胰岛素原中加入柠康酐。
在一种实施方式中，所述步骤(4)通过胰蛋白酶酶切转化修饰后的含有权利要求1所述序列的甘精胰岛素原，去除所述的融合肽，得到B 29位赖氨酸被柠康酐残基修饰的甘精胰岛素。
在一种实施方式中，所述步骤(4)在pH1.5～2.5酸性水解除去柠康酐残基，得到甘精胰岛素。
在一种实施方式中，所述步骤(5)使用离子交换色谱法纯化甘精胰岛素，以获得高纯度的甘精胰岛素；
在一种实施方式中，所述步骤(5)使用制备型HPLC色谱进一步纯化甘精胰岛素以获得更高纯度的甘精胰岛素；
在一种实施方式中，还对高纯度的甘精胰岛素经过结晶和干燥形成最终的甘精胰岛素原料药。
在一种实施方式中，将上述优化的基因连接到合适的载体，如pTAC表达质粒系列、pGEX系列或pET系列，优选pET系列质粒，更优选质粒pET-28a；该质粒可转染K12 JM109工程菌或K12 W110工程菌以形成表达克隆。在另一个优选的实施方案中，表达质粒转染到BL21(DE3)工程菌中。
在一种实施方式中，所述重组大肠杆菌通过摇瓶或发酵罐培养至适当浓度，然后诱导甘精胰岛素原的表达。
在一种实施方式中，将含有甘精胰岛素原的包涵体的细胞通过溶菌酶处理和高压均质化进行裂解；分离的包涵体由含有洗涤剂或低浓度离液剂的溶液洗涤，并用高pH缓冲溶液溶解。
在一个实施方式中，溶解缓冲液的pH值为11.6-12.4，溶解缓冲液含有Tris、EDTA和L-半胱氨酸；所述Tris的浓度为10-50mM，EDTA的浓度为0.05-1.0mM，L-半胱氨酸的浓度为0.25-5.0mM。
在一个实施方式中，Tris的浓度为20-30mM；EDTA的浓度为0.05-0.25mM，L-半胱氨酸的浓度为0.25-1.0mM；pH为11.8-12.2。
在一种实施方式中，溶解缓冲液的温度为10-30℃，或15-25℃；包涵体溶解时间为10-120min，或10-60min。
在一个实施方式中，溶解缓冲液的pH值为10.0-11.6，或为10.8-11.4；溶液的温度约10-25℃，或15-20℃；总蛋白的浓度为1-10g/L，或1-7g/L；复性持续时间为12-48h，或为24-36h。
在一种实施方式中，酶切前采用保护试剂修饰甘精胰岛素原；所述保护试剂可以是易于与B29-Lys的γ-NH 2基团反应的亲电试剂，例如酸酐，包括但不限于乙酸酐，柠檬酸酐或柠康酐。
在一个实施方式中，向甘精胰岛素原中加入过量摩尔比的柠康酐；可选地，对于甘精胰岛素原，加入10倍或更多摩尔量的柠康酐；或加入20倍或更多摩尔量的柠康酐。
在一个实施方式中，加入保护试剂修饰的反应温度为15-25℃，pH为8.0-9.0，持续时间2-8h。
在一种实施方式中，修饰完成后可加入乙醇胺中和过量的柠康酐，乙醇胺的体积为柠康酐的40％-80％，终止时间为10-30min。
在一个实施方式中，将复性溶液的蛋白浓度调节为1-7g/L。向复性溶液中加入按体积计 0.2％柠康酐并将pH调节至8.5，在20℃下反应4小时；修饰完成后，加入修饰所用柠康酐体积60％的乙醇胺进行中和，pH调节为9.0，中和30min。将终浓度为220U/g蛋白的胰蛋白酶(优选牛胰蛋白酶)加入到柠康酸修饰的甘精胰岛素原中，将pH调节至9.0，并在20℃下消化24小时以获得具有柠康酸残基的甘精胰岛素。通过HPLC-RP(C18)监测该过程。一旦酶切反应结束，通过加入盐酸并调节pH 2.0-2.5可以终止酶切反应。让溶液在该低pH值下保持24小时，以水解B29-Lys上的柠康酸残基，得到甘精胰岛素。
在一种实施方式中，将复性液的蛋白浓度调节为4g/L后，直接向复性的蛋白质溶液中加入按体积计0.05％柠康酐，将pH调节至8.5，在20℃下反应2小时。修饰完成后，加入修饰所用柠康酐体积的60％的乙醇胺进行中和，pH调节为9.4，中和15min。然后将终浓度为0.063mg/g蛋白的胰蛋白酶加入到柠康酸修饰的甘精胰岛素中，将pH调节至9.0，并在20℃下反应24小时以获得具有柠康酸残基的甘精胰岛素。通过HPLC-RP(C18)监测该过程。一旦酶切反应完成，通过加入盐酸并调节pH 2.0-2.5以终止反应。让溶液在该低pH值下保持12小时，通过水解去除B29-Lys上的柠康酸残基，得到甘精胰岛素。
在一种实施方式中，在胰蛋白酶酶解后，加入终浓度为1-10mM的锌离子，并且调节pH为5.5-6.5，使得甘精胰岛素形成沉淀并析出，通过适当的方法对甘精胰岛素进行纯化，从而获得最终甘精胰岛素产品。
在一种实施方式中，使用制备的RP-HPLC与磷酸二氢铵缓冲体系进行一步纯化，其中磷酸氢二铵的浓度为0.05-0.3M，或0.05-0.2M；pH值在2.0-5.0的范围内；有机改性剂可以是乙醇，甲醇或乙腈；在有机溶剂的线性浓度梯度中，甘精胰岛素被洗脱。
在一种实施方式中，使用制备的RP-HPLC和Tris-HCl缓冲体系进行最终纯化，其中Tris浓度为0.02-0.3M，或0.02-0.2M；pH值在7.0-9.0的范围内；有机改性剂可以是乙醇，甲醇或乙腈；在有机溶剂的线性浓度梯度中，甘精胰岛素被洗脱。
在一种实施方式中，从RP-HPLC洗脱的甘精胰岛素利用等电点沉淀法进行沉淀，收集沉淀。收集到的沉淀用0-1.5％的氯化钠溶液进行重悬洗涤，并将洗涤后离心得到的湿固体用40-200mM的盐酸溶液进行溶解，并且将甘精胰岛素的浓度调节为20-40mg/mL，调节pH至3.0-5.0，过滤后滤液冷冻干燥，制备获得以晶体或固化的API形式存在的甘精胰岛素原料药。
本发明还要求保护所述方法在制备甘精胰岛素或含甘精胰岛素的药物中的应用。
有益效果：
本发明以定点突变后的SOD片段为融合肽并采用“0C肽”策略，可以获得较高发酵产量，将甘精胰岛素的发酵产率提高了75％及以上，最高提高了78％；
本发明通过“0C肽”策略，避免了C肽残基的残留，减少酶转化步骤的质量损失，本专 利申请中减少错误的重折叠和错误的酶切过程造成的杂质，提高最终产品的产量和纯度，主峰色谱纯度达到99.4以上，最大单杂≤0.16％，其中较佳实施例中主峰色谱纯度高达99.9％，最大单杂控制在0.05％以下；主峰色谱纯度相对对比例提高了7％，最大单杂含量相对对比例由2.15％降低到≤0.16％，明显降低一个数量级。
本发明的制备方法中通过牛胰蛋白酶进行酶切处理，相比于猪胰蛋白酶，酶切转化率可以提高16％。因此，制备高质量甘精胰岛素的生产成本可以大大降低。
附图说明
图1是根据本发明的具体实例6中，采用基因序列SEQ ID NO:11构建后的菌株纯化液相色谱图。
图2是根据本发明的具体实例7中，采用基因序列SEQ ID NO:12构建后的菌株纯化液相色谱图。
图3是根据本发明的具体实例8中，采用基因序列SEQ ID NO:13构建后的菌株纯化液相色谱图。
图4A、4B和4C是根据本发明的具体实例1-3中，采用基因序列SEQ ID NO:10构建后的菌株WCB01复性后的液相色谱图谱。
图5A、5B和5C是根据本发明的具体实例1-3中，采用基因序列SEQ ID NO:15构建后的菌株WCB02复性后的液相色谱图谱。
图6是根据本发明的具体实例1-3中，采用基因序列SEQ ID NO:16构建后的菌株WCB03复性后的液相色谱图谱。
图7是根据本发明的具体实例1-3中，采用基因序列SEQ ID NO:17构建后的菌株WCB04复性后的液相色谱图谱。
图8是根据本发明的具体实例1-3中，采用基因序列SEQ ID NO:18构建后的菌株WCB05复性后的液相色谱图谱。
图9A是根据本发明的具体实例1-5中，采用基因序列SEQ ID NO:10构建后的菌株WCB01纯化后的液相色谱图谱。
图9B是根据本发明的具体对比例1-3中，采用基因序列SEQ ID NO:15构建后的菌株WCB02纯化后的液相色谱图谱。
图10A是根据本发明的具体实例4中，采用牛胰蛋白酶对甘精胰岛素原的酶切效果的液相色谱图谱。
图10B是根据本发明的具体对比例4中，采用猪胰蛋白酶对甘精胰岛素原的酶切效果的液相色谱图谱。
具体实施方式
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：新甘精胰岛素原的结构设计
设计如式I所示的甘精胰岛素原蛋白序列：
R-R 1-(B 1-B 32)-(A 1-A 20)-A 21
改良后的甘精胰岛素原的序列采用“0C肽”策略，即B链和A链间没有氨基酸序列。N端先导氨基酸序列可以增强表达，保护甘精胰岛素原，防止被大肠杆菌降解。其中，融合肽R-R 1中R的氨基酸序列为MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP(如SEQ ID NO:3所示)。该融合肽氨基酸序列的C端通过精氨酸或赖氨酸残基连接到甘精胰岛素的B链，最终融合肽通过胰蛋白酶裂解被除去。
实施例2：构建含有新甘精胰岛素原编码基因的重组质粒
按照实施例1的方法设计新甘精胰岛素原的序列：新甘精胰岛素原中(B 1-B 32)-(A 1-A 20)-A 21的序列为SEQ ID NO:6。其中，甘精胰岛素原带融合肽的全序列为SEQ ID NO:10。为了确保在大肠杆菌中有效表达SEQ ID NO:10所示的融合蛋白质，对遗传密码子进行了优化。优化后的基因序列如SEQ ID NO:14所示，其含有一个5’NcoI位点(CCATGG)和1个3’Hind III位点(AAGCTT)。
通过商业CRO公司化学合成SEQ ID NO：14的DNA片段，将该DNA片段用NcoI和Hind III限制酶裂解，插入到用相同的限制性内切酶裂解的pET-28a表达载体中，通过连接酶连接，形成pET-PIG-1表达载体。
实施例3：构建表达新甘精胰岛素原的重组大肠杆菌
将实施例2构建的的重组表达载体pET-PIG-1转染到大肠杆菌BL21(DE3)感受态细胞中。通过卡那霉素抗性筛选阳性克隆并用DNA测序进行确认。对阳性克隆在37℃温度条件下进行培养和扩增，然后无菌培养基和甘油加入细胞中。将1mL的细胞培养液转入无菌的安瓿中，并在-80℃进行保存，形成甘精胰岛素原工作种子库(WCB01)。
实施例4：甘精胰岛素原融合蛋白的表达
将实施例3得到的WCB01按0.2％的接种量接种于MLB培养基(含15g/L酵母粉和5g/L氯化钠)中，37℃、250rpm条件下培养6-14h，得到一级种子液。将一级种子液按0.2％的接种量接种于BFM培养基(含6g/L磷酸氢二铵，4g/L氯化铵，13.5g/L磷酸二氢钾，1.39g/L七水硫酸镁，2.8g/L一水柠檬酸，8g/L葡萄糖，3g/L酵母粉和1mL/L微量元素溶液(含10g/L七水合硫酸亚铁，1.1g/L氯化锌，1.0g/L五水合硫酸铜，0.4g/L四水合氯化 锰，0.2g/L硼酸，2.7g/L氯化钙和0.2g/L钼酸钠)进一步培养8-16h得到二级种子液。然后按照体积比1：10接种到发酵罐BFM培养基中，生长温度30-39℃、生长溶氧10-50％和生长pH6.0-7.3条件下连续培养12-18h，直到发酵液的OD 600为100-200，往发酵罐中加入终浓度为0.1-1.0mM IPTG诱导甘精胰岛素原进行表达，其他生长条件不变。连续诱导8-16h，通过离心机收集菌体。
将含有甘精胰岛素原的包涵体的菌体用25mM Tris，10mM EDTA，pH为8.0的缓冲液进行重悬，控制菌体浓度为200g/L。菌体通过溶菌酶处理和高压均质进行裂解，对菌体裂解液进行离心收集包涵体沉淀，去除上清。
用25mM Tris，1M尿素，1％吐温20，pH为8.0的洗涤液对包涵体进行洗涤。洗涤完成后，用25mM Tris，0.1mM EDTA和0.5mM L-半胱氨酸缓冲液重悬包涵体，调节pH为12.0，在15℃下溶解50min。溶解后的溶液命名为包涵体溶解液。
实施例5：甘精胰岛素原的复性
将实施例4制备的WCB01的包涵体溶解液用1μm的PP滤芯过滤，将温度控制为20℃，调节pH为11.0，然后进行复性32h得到复性的甘精胰岛素原。
实施例6：酶切转化和纯化制备甘精胰岛素
(一)酶切制备甘精胰岛素
复性完成后，加入复性液按体积计0.2％的柠康酐进行修饰。调节复性液的pH为8.5，搅拌修饰2h。修饰完成后，添加修饰所用的柠康酐量60％的乙醇胺中和过量的柠康酐，pH调节为9.4，中和15min。然后直接加入终浓度为0.063mg牛胰蛋白酶/g蛋白的牛胰蛋白酶，调节pH为9.0，在20℃下酶切24h。去除融合肽，获得带柠康酐修饰的甘精胰岛素。酶切工程用RP-HPLC(C18)进行监控。当酶切结束后，通过盐酸调节pH为2.0终止酶切反应。并在pH2.0下保持12h，水解B 29位被柠康酐修饰的赖氨酸，获得甘精胰岛素。水解完成后，加入终浓度为3mM的氯化锌，调节pH为6.0，使甘精胰岛素形成絮状沉淀。
(2)甘精胰岛素的纯化
将步骤(1)酶切和水解后的甘精胰岛素沉淀用体积分数为3％的乙酸进行溶解，溶解pH值为3.5。溶解的甘精胰岛素作为样品加载到阳离子层析柱上，用缓冲液平衡。甘精胰岛素可以被30％的异丙醇，1.0M氯化钠线性梯度洗脱。阳离子层析纯化后，用终浓度为3mM的氯化锌，调节pH 7.3，使甘精胰岛素形成絮状沉淀。再重复上述操作2次。
将阳离子层析纯化的甘精胰岛素加载到反相制备层析柱上。用0.1M磷酸二氢铵与乙腈按照9:1混合后调节pH为3.5得到的溶液平衡层析柱。洗脱缓冲液是pH为3.5的0.1M磷酸氢二铵和10％乙腈的混合溶液与60％的乙腈按照不同比例混合后得到的溶液。甘精胰岛素通 过洗脱缓冲液的线性梯度洗脱下来。获得的甘精胰岛素洗脱液的中甘精胰岛素的纯度为97％。第一次反相层析纯化后，用终浓度为3mM的氯化锌，调节pH 7.3，使甘精胰岛素形成絮凝沉淀。甘精胰岛素沉淀用3％的乙酸进行溶解，溶解pH值为3.5。溶解的甘精胰岛素作为样品加载到反相制备层析柱上。用0.05M Tris与乙腈按照9:1混合后调节pH为8.5得到的溶液平衡层析柱。洗脱缓冲液是pH为8.5的0.05M Tris和10％乙腈的混合溶液与60％乙腈按照不同比例混合得到的溶液。甘精胰岛素通过洗脱缓冲液的线性梯度洗脱下来，经测定，甘精胰岛素洗脱液中的甘精胰岛素纯度为99.9％。第二次反相层析纯化后，用100mM的盐酸溶液调节pH 7.3，甘精胰岛素形成絮凝沉淀。将收集的沉淀物用0.3％氯化钠溶液(pH 7.0)重悬洗涤3次，离心得到甘精胰岛素湿固体。
实施例7甘精胰岛素原料药的制备
用100mM盐酸水溶液溶解实施例6制备的甘精胰岛素湿固体，直至浓度为30mg/mL，调节pH 4.0，用0.22μmPES滤膜过滤，将过滤后的甘精胰岛素溶液转移至冷冻干燥机中，冷冻干燥程序的设定参数如下：
1)进料前搁板制冷：设定温度-30℃；
2)制冷控制：设定温度-30℃，设定时间240min，持续时间240min；
3)捕水器制冷：设定温度-50℃，持续时间为10min；
4)预抽真空：预抽真空0.2000mbar，设定报警真空0.5000mbar，报警真空持续时间10s；
5)一次干燥：设定温度-10℃，设定时间240min，持续时间3600min，设定真空0.1800mbar；
6)解析干燥：设定温度25℃，设定时间480min，持续时间240min，设定真空0.1800mbar。
经上述处理后得到最终的甘精胰岛素原料药，纯度为99.9％以上。
实施例8甘精胰岛素的制备
具体实施方式同实施例2～6，区别在于，甘精胰岛素原带融合肽的全序列如SEQ ID NO:7所示，编码该甘精胰岛素原带融合肽的基因序列如SEQ ID NO:11所示。构建的重组大肠杆就在相同条件下发酵生产的甘精胰岛素原融合蛋白表达量为5.8g/L，纯化后的产品经HPLC检测，获得99.42％的高主峰色谱纯度以及0.16％的最大单杂色谱纯度(具体见图1和表1)。
表1为色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 11.226 12.93 0.16
2 13.513 0.79 0.01
3 16.309 3.22 0.04
4 16.792 1.10 0.01
5 17.316 3.26 0.04
6 17.887 4.05 0.05
7 18.525 2.18 0.03
8 18.937 4.83 0.06
9 19.675 7828.73 99.42
实施例9甘精胰岛素的制备
具体实施方式同实施例2～6，区别在于，甘精胰岛素原带融合肽的全序列如SEQ ID NO:8所示，编码该甘精胰岛素原带融合肽的基因序列如SEQ ID NO:12所示。构建的重组大肠杆就在相同条件下发酵生产的甘精胰岛素原融合蛋白表达量为6.1g/L，纯化后的产品经HPLC检测，获得99.59％的高主峰色谱纯度以及0.14％的最大单杂色谱纯度(具体见图2和表2)。
表2色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 20.927 1134 0.02
2 21.789 6077592 99.59
3 22.388 4444 0.07
4 23.208 1540 0.03
5 25.206 8487 0.14
6 28.111 1759 0.03
7 28.604 2850 0.05
8 28.897 321 0.01
9 29.017 318 0.01
10 29.091 1534 0.03
11 29.392 1147 0.02
12 29.877 1119 0.02
13 30.001 602 0.01
实施例10甘精胰岛素的制备
具体实施方式同实施例2～6，区别在于，甘精胰岛素原带融合肽的全序列如SEQ ID NO:9所示，编码该甘精胰岛素原带融合肽的基因序列如SEQ ID NO:13所示。构建的重组大肠杆就在相同条件下发酵生产的甘精胰岛素原融合蛋白表达量为6.0g/L，纯化后的产品经HPLC检测，获得99.80％的高主峰色谱纯度以及0.09％的最大单杂色谱纯度(具体见图3和表3)。
表3色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 18.410 6105 0.09
2 17.715 1916 0.03
3 21.611 6832023 99.80
4 28.367 1690 0.02
5 29.131 2405 0.04
6 29.849 813 0.01
7 29.999 737 0.01
对比例1：构建未经优化的甘精胰岛素原的重组大肠杆菌
具体实施方式同实施例2～3，区别在于，将融合肽R-R 1中的氨基酸序列替换为MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPR(SEQ ID NO:1)，编码SEQ ID NO:1的整个氨基酸序列的DNA片段相应调整为：
Figure PCTCN2020138950-appb-000003
使甘精胰岛素原带融合肽的全序列为MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPKFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG(SEQ ID NO:15)；
然后用NcoI和Hind III限制酶将编码上述氨基酸序列的核苷序列与质粒pET-28a酶切处 理，将酶切后的片段与载体连接，获得重组表达载体pET-PIG-2。
将重组表达载体pET-PIG-2转化至大肠杆菌BL21(DE3)感受态细胞中。通过卡那霉素抗性筛选阳性克隆并用DNA测序进行确认。对阳性克隆进行培养和扩增，然后无菌培养基和甘油加入细胞中。将1mL的细胞培养液转入无菌的安瓿中，并在-80℃进行保存，形成甘精胰岛素原工作种子库(WCB02)。
对比例2：甘精胰岛素原融合蛋白的表达
将对比例1得到的重组菌WCB02按照实施例4所述方法进行三批次的培养，并处理得到包涵体溶解液，随后按照实施例5所述方法进行复性。利用HPLC分别检测通过发酵WCB01和WCB02得到的甘精胰岛素原复性前体的产量，三组平行实验的结果如表4所示。WCB01复性液相色谱图谱如图4A、4B和4C所示，相关数据如表4A、4B和4C所示；WCB02复性液相色谱图谱如图5A、5B和5C所示，相关数据如表5A、5B和5C所示。
表4不同甘精胰岛素序列表达后甘精胰岛素原复性前体产量
Figure PCTCN2020138950-appb-000004
表1结果显示，使用定点突变的SOD片段作为融合肽以及采用“0C肽”策略的优选序列SEQ ID NO:10，可以获得更有效的表达以及更为稳定的高发酵产量，具体是将甘精胰岛素的发酵产率提高了75％及以上，最高提高了78％。
表4A色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 5.981 3728 0.02
2 6.670 2863 0.02
3 7.336 96294 0.62
4 8.800 30923 0.20
5 9.653 63346 0.41
6 9.981 14592 0.09
7 10.447 15349 0.10
8 11.440 95758 0.62
9 11.877 132292 0.86
10 12.207 217422 1.41
11 13.066 6246235 40.40
12 13.486 2272194 14.69
13 14.046 2436344 15.76
14 14.868 472685 3.06
15 15.527 370660 2.40
16 16.669 658939 4.26
17 17.230 168242 1.09
18 17.739 917577 5.93
19 18.501 735984 4.76
20 19.546 270981 1.75
21 20.324 141662 0.92
22 21.023 98337 0.64
表4B色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.295 2536 0.02
2 7.619 1584 0.01
3 7.643 1159 0.01
4 8.441 24235 0.20
5 8.945 1332 0.01
6 9.322 46100 0.37
7 9.671 18693 0.15
8 10.116 12874 0.10
9 10.464 6401 0.05
10 11.134 100618 0.82
11 11.542 107712 0.87
12 11.924 84129 0.68
13 12.079 72595 0.59
14 12.838 6335431 51.42
15 13.381 1267742 10.29
16 13.822 1891466 15.35
17 14.168 547291 4.44
18 14.719 330588 2.68
19 15.360 275838 2.24
20 15.856 33956 0.28
21 16.134 93890 0.76
22 16.482 398809 3.24
23 16.969 95196 0.77
24 17.577 397227 3.22
25 18.013 54857 0.45
26 18.401 119467 0.97
表4C色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.290 2542 0.02
2 7.614 1385 0.01
3 7.642 1119 0.01
4 8.405 26003 0.20
5 8.947 1558 0.01
6 9.338 49743 0.38
7 9.676 21486 0.16
8 10.137 11248 0.09
9 10.509 6231 0.05
10 11.161 80814 0.62
11 11.552 115175 0.88
12 11.919 201458 1.54
13 12.833 6554868 50.05
14 13.352 1500343 11.46
15 13.827 2051646 15.67
16 14.165 580765 4.43
17 14.695 409681 3.13
18 15.405 186479 1.42
19 15.716 133971 1.02
20 16.110 89273 0.68
21 16.466 367821 2.81
22 16.971 120239 0.92
23 17.569 353694 2.70
24 17.854 50995 0.39
25 17.973 54941 0.42
26 18.382 121957 0.93
表5A色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.153 43402 0.50
2 8.547 10401 0.12
3 9.409 28985 0.34
4 10.141 3161 0.04
5 11.207 31408 0.37
6 11.584 59171 0.69
7 11.962 47234 0.55
8 12.828 3525420 40.98
9 13.249 1256739 14.61
10 13.820 1511732 17.57
11 14.646 279064 3.24
12 15.240 205973 2.39
13 16.454 370498 4.31
14 17.510 552834 6.43
15 18.300 264290 3.07
16 18.893 119583 1.39
17 19.338 137776 1.60
18 20.046 88628 1.03
19 20.821 42519 0.49
20 21.517 23344 0.27
表5B色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.105 44323 0.44
2 8.487 12383 0.12
3 9.382 25154 0.25
4 9.738 4008 0.04
5 10.096 5892 0.06
6 10.462 3146 0.03
7 11.132 39277 0.39
8 11.532 51520 0.51
9 11.867 48987 0.49
10 12.749 3568760 35.41
11 13.174 1772217 17.58
12 13.732 1090494 10.82
13 14.101 417898 4.15
14 14.555 277829 2.76
15 15.211 175373 1.74
16 16.366 535098 5.31
17 17.420 472387 4.69
18 17.448 387633 3.85
19 18.199 539587 5.35
20 19.199 410304 4.07
21 20.267 87937 0.87
22 20.763 109568 1.09
表5C色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.197 2058 0.03
2 7.586 960 0.01
3 7.627 1039 0.01
4 8.390 17399 0.22
5 8.935 1431 0.02
6 9.296 30156 0.38
7 9.673 23883 0.30
8 10.159 6689 0.09
9 10.513 4025 0.05
10 10.864 2450 0.03
11 11.120 39285 0.50
12 11.560 64445 0.82
13 11.918 141948 1.81
14 12.827 3726812 47.46
15 13.260 1169681 14.90
16 13.830 1192710 15.19
17 14.172 363760 4.63
18 14.667 241046 3.07
19 15.381 102318 1.30
20 15.673 45485 0.58
21 15.927 4970 0.06
22 16.170 36141 0.46
23 16.509 215523 2.74
24 16.941 45912 0.58
25 17.322 32154 0.41
26 17.582 166907 2.13
27 17.837 67783 0.86
28 18.439 104829 1.34
对比例3含有1个突变的融合肽序列的甘精胰岛素原的表达
调整实施例1的策略设计编码甘精胰岛素的序列，并在宿主细胞中表达，使甘精胰岛素原含有在SEQ ID NO：15的基础上进行了1个定点突变的融合肽R序列，氨基酸序列设计为：
MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG(SEQ ID NO:16)；编码该氨基酸序列的核苷酸序列如SEQ ID NO:19所示；
按照实施例5中所述方法进行复性。利用HPLC检测菌株发酵得到的甘精胰岛素原复性 前体的产量，结果显示，相同条件下的甘精胰岛素原复性前体产量为1.9g/L。复性液相色谱图谱分别如图6所示，相关数据如表6所示。
表6色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.100 31530 0.58
2 8.481 6966 0.13
3 9.372 14681 0.27
4 9.756 2109 0.04
5 10.125 3795 0.07
6 10.475 3608 0.07
7 11.172 25035 0.46
8 11.546 42006 0.77
9 11.889 77271 1.42
10 12.789 1939000 35.64
11 13.211 814935 14.98
12 13.775 613260 11.27
13 14.144 231346 4.25
14 14.573 160525 2.95
15 15.227 96528 1.77
16 16.406 305068 5.61
17 17.445 459176 8.44
18 18.186 286812 5.27
19 19.207 180833 3.32
20 19.945 50905 0.94
21 20.343 48036 0.88
22 20.867 30012 0.55
23 21.435 15293 0.28
24 22.233 1649 0.03
对比例4含有2个突变的融合肽序列的甘精胰岛素原的表达
调整实施例1的策略设计编码甘精胰岛素的序列，并在宿主细胞中表达，使甘精胰岛素原含有在SEQ ID NO：15的基础上进行了2个定点突变的融合肽R序列，氨基酸序列分别设计为：
MATKAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG(SEQ ID NO:17)；编码该氨基酸序列的核苷酸序列如SEQ ID NO:20所示；
按照实施例5中所述方法进行复性。利用HPLC分别检测菌株发酵得到的甘精胰岛素原复性前体的产量，结果显示，相同条件下的甘精胰岛素原复性前体产量为2.2g/L。复性液相色谱图谱如图7所示，相关数据分别如表7所示。
表7色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.080 32442 0.54
2 8.429 7111 0.12
3 9.337 17594 0.29
4 10.104 1634 0.03
5 11.042 10294 0.17
6 11.485 36434 0.61
7 11.850 108086 1.80
8 12.720 2197231 36.59
9 13.132 1050997 17.50
10 13.712 931607 15.51
11 14.506 172841 2.88
12 15.214 174204 2.90
13 16.344 273268 4.55
14 17.415 359878 5.99
15 17.744 148993 2.48
16 18.227 137118 2.28
17 18.752 106493 1.77
18 19.230 109246 1.82
19 19.905 82369 1.37
20 20.807 25772 0.43
21 21.341 21575 0.36
对比例5含有C肽的甘精胰岛素原的表达
调整实施例1的策略设计编码甘精胰岛素的序列，并在宿主细胞中表达，使甘精胰岛素原含有“C肽”(EAR)，氨基酸序列设计为：
MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREARGIVEQCCTSICSLYQLENYCG(SEQ ID NO:18)；编码该氨基酸序列的核苷酸序列如SEQ ID NO:21所示.
按照实施例5中所述方法进行复性。利用HPLC检测菌株发酵得到的甘精胰岛素原复性前体的产量，结果如下表8所示。复性液相色谱图谱如图8所示，相关数据如表9所示。
表8不同甘精胰岛素序列表达后甘精胰岛素原复性前体产量
Figure PCTCN2020138950-appb-000005
表6结果显示，使用定点突变1个或者2个位点的融合肽序列以及采用“C肽”(REA)策略的融合肽序列获得的发酵产量远远低于WCB01的产量
表9色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 7.094 30377 0.57
2 8.487 7761 0.15
3 9.382 14594 0.27
4 9.743 2603 0.05
5 10.105 4467 0.08
6 10.482 3140 0.06
7 11.115 25287 0.48
8 11.541 33463 0.63
9 11.862 75536 1.42
10 12.763 1878930 35.33
11 13.179 848037 15.95
12 13.740 611936 11.51
13 14.113 245888 4.62
14 14.557 172803 3.25
15 15.205 112415 2.11
16 16.377 259256 4.87
17 17.439 418883 7.88
18 18.243 305379 5.74
19 19.245 131360 2.47
20 20.040 73644 1.44
21 20.787 59947 1.13
对比例6：复性、酶切转化和纯化
将对比例2制备的包涵体溶解液分别按照实施例5的条件进行复性。然后按照实施例6中的方法对复性后的样品进行修饰、酶切以及纯化。重组菌WCB01和对比例2的重组菌WCB02的样品纯化后的液相色谱图谱分别如图9A和图9B所示，相关数据分别如表10A和10B所示。结果显示，使用定点突变3个氨基酸的SOD片段作为融合肽以及采用“0C肽”策略的菌株(WCB01)，可以获得99.93％的高主峰色谱纯度以及0.05％的最大单杂色谱纯度。与此同时，使用未突变的SOD片段作为融合肽的菌株(WCB02)的主峰和最大单杂的色谱纯度分别为92.25％和2.15％。使用突变的SOD片段以及采用“0C肽”策略，虽然主峰纯度提高了一点，但是最大单杂含量明显降低一个数量级，避免C-肽残基的残留，减少酶切转化的质量损失和错切杂质。
表10A色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 19.960 6005658 99.93
2 21.681 3011 0.05
3 29.279 1181 0.02
表10B色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 6.450 753 0.01
2 7.657 420 0.01
3 80184 609 0.01
4 8.438 538 0.01
5 8.510 268 0.00
6 8.576 256 0.00
7 8.709 792 0.01
8 8.816 316 0.00
9 8.879 360 0.00
10 8.972 987 0.01
11 9.162 449 0.01
12 9.186 268 0.00
13 9.304 365 0.00
14 9.410 1019 0.01
15 9.598 450 0.01
16 9.694 625 0.01
17 9.804 741 0.01
18 9.880 559 0.01
19 10.156 2983 0.04
20 10.475 739 0.01
21 11.001 119807 1.49
22 11.531 2151 0.03
23 12.047 1591 0.02
24 12.292 1474 0.02
25 12.465 2309 0.03
26 12.752 14885 0.18
27 12.949 15308 0.19
28 13.220 17592 0.22
29 13.536 52838 0.66
30 13.878 85586 1.06
31 14.381 8191 0.10
32 15.065 10546 0.13
33 16.135 8074 0.10
34 16.763 173201 2.15
35 17.114 21542 0.27
36 17.819 1731 0.02
37 19.010 20447 0.25
38 19.736 7428281 92.25
39 20.859 50936 0.63
40 22.476 53 0.00
41 22.666 1348 0.02
42 23.631 109 0.00
43 23.721 434 0.01
44 29.584 191 0.00
对比例7：利用猪胰蛋白酶的酶切转化
按照实施例4中的酶切方法利用猪胰蛋白酶对实施例3制备的WCB01复性后的溶液进行酶切转化。分别利用猪胰蛋白酶和牛胰蛋白酶的酶切转化的液相色谱图谱分别如图10A和图10B所示，相关数据分别如表11A和11B所示。图10A中出峰时间为20.447min的主峰面积为9569627mAU*min，图10B中出峰时间为20.730min的主峰面积为11142487mAU*min。结果显示，使用牛胰蛋白酶相对猪胰蛋白酶，酶切转化率可以提高16％；因为产物浓度与峰面积存在正比关系，转化率根据如下公式计算得到(11142487-9569627)/9569627＝16％。
表11A色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 6.123 25974 0.09
2 6.576 32496 0.12
3 7.150 18545 0.07
4 7.344 2774 0.01
5 7.593 36611 0.13
6 7.897 26753 0.10
7 8.428 65286 0.23
8 8.607 16521 0.06
9 8.825 15502 0.06
10 9.011 92535 0.33
11 9.435 27660 0.10
12 9.628 10516 0.04
13 9.787 5496 0.02
14 9.894 5612 0.02
15 10.066 9195 0.03
16 10.212 16404 0.06
17 10.423 6520 0.02
18 10.570 39115 0.14
19 10.880 29368 0.10
20 11.451 70108 0.25
21 12.002 3610530 12.83
22 12.403 98065 0.35
23 12.783 20620 0.07
24 130.84 125933 0.45
25 13.728 31204 0.11
26 14.421 134964 0.48
27 15.341 58382 0.21
28 15.609 18801 0.07
29 16.793 115174 0.41
30 17.311 2486 0.04
31 17.509 12968 0.05
32 18.124 111013 0.39
33 18.809 115239 0.41
34 19.372 70992 0.25
35 20.447 9569327 34.00
36 21.527 2327425 8.27
37 22.216 5483055 19.48
38 23.270 395684 1.41
39 24.581 2141960 7.61
40 24.992 611732 2.17
41 26.320 440323 1.56
42 26.819 2029210 7.21
43 28.805 61812 0.22
44 29.604 4203 0.01
表11B色谱检测到的主要峰面积及保留时间
# 保留时间 峰面积 ％峰面积
1 6.141 96963 0.29
2 6.648 41805 0.13
3 7.202 38587 0.12
4 7.374 14189 0.04
5 7.629 21517 0.06
6 7.928 54454 0.16
7 8.446 73781 0.22
8 8.653 45482 0.14
9 8.868 21845 0.07
10 9.061 100605 0.30
11 9.229 18851 0.06
12 9.486 30588 0.09
13 9.675 14226 0.04
14 9.821 7601 0.02
15 9.973 11309 0.03
16 10.093 8934 0.03
17 10.252 18843 0.06
18 10.435 6463 0.02
19 10.618 37421 0.11
20 10.929 24440 0.07
21 11.504 79186 0.24
22 12.104 475734 1.42
23 12.472 78628 0.24
24 12.870 33249 0.10
25 13.180 160861 0.48
26 13.862 35587 0.11
27 14.570 76881 0.23
28 14.817 73991 0.22
29 15.508 75852 0.23
30 16.301 20875 0.06
31 17.032 106983 0.32
32 17.735 29030 0.09
33 18.272 198861 0.60
34 18.860 131756 0.39
35 19.604 78034 0.23
36 20.730 11142487 33.37
37 21.832 2758566 8.26
38 22.481 9109465 27.28
39 23.599 505312 1.51
40 24.919 2404115 7.20
41 25.339 1005576 3.01
42 27.172 4038121 12.09
43 29.079 87112 0.26
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种甘精胰岛素原，其特征在于，含有如下所示结构的氨基酸序列：
    (B 1-B 32)-(A 1-A 20)-A 21；
    其中：
    B 1-B 32为在天然人胰岛素的B链B 1-B 30的B 30位C端后连接了两个精氨酸Arg残基；
    A 1-A 20为具有20个氨基酸的胰岛素A链；
    A 21是甘氨酸。
  2. 根据权利要求1所述的甘精胰岛素原，其特征在于，氨基酸序列的结构为：
    R-R 1-(B 1-B 32)-(A 1-A 20)-A 21；其中：
    R-R 1为融合肽序列，R的氨基酸序列为：MATX 1AVSVLKGDGPVQGIINFEQX 2ESNGPVKVWGSIX 3GLTEGLHGFHVHEFGDNTAGSTSAGP；X 1为脯氨酸或组氨酸；X 2为脯氨酸或组氨酸；X 3为脯氨酸或组氨酸；
    R 1为精氨酸或赖氨酸。
  3. 根据权利要求1所述的甘精胰岛素原，其特征在于，R的氨基酸序列如SEQ ID NO:2或SEQ ID NO:3所示。
  4. 编码权利要求1～3任一所示甘精胰岛素原的DNA。
  5. 含有权利要求4所述DNA的表达载体。
  6. 表达权利要求1～3任一所述甘精胰岛素原或含有权利要求5所述表达载体的非植物细胞。
  7. 一种生产甘精胰岛素的方法，其特征在于，将表达权利要求1～3任一所述甘精胰岛素原的重组大肠杆菌于35～37℃下发酵至少20h，生产甘精胰岛素。
  8. 根据权利要求7所述的方法，其特征在于，还对发酵后的甘精胰岛素酶切、修饰、复性和纯化。
  9. 根据权利要求8所述的方法，其特征在于，所述酶切采用胰蛋白酶进行酶切；所述修饰采用柠康酐。
  10. 权利要求7～9任一所述方法在制备甘精胰岛素或含甘精胰岛素的药物中的应用。
PCT/CN2020/138950 2020-09-11 2020-12-24 一种新甘精胰岛素原及其制备甘精胰岛素的方法 Ceased WO2022052368A1 (zh)

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