WO2020051812A1 - 一种新型门冬胰岛素原的结构和制备门冬胰岛素的方法 - Google Patents

一种新型门冬胰岛素原的结构和制备门冬胰岛素的方法 Download PDF

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WO2020051812A1
WO2020051812A1 PCT/CN2018/105314 CN2018105314W WO2020051812A1 WO 2020051812 A1 WO2020051812 A1 WO 2020051812A1 CN 2018105314 W CN2018105314 W CN 2018105314W WO 2020051812 A1 WO2020051812 A1 WO 2020051812A1
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sequence
insulin
seq
aspart
proinsulin
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PCT/CN2018/105314
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French (fr)
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汤传根
潘尚书
刘晓锐
李宬
崔怀言
陈松
张昊宁
丁捷飞
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美药星（南京）制药有限公司
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Priority to CN201880090551.8A priority Critical patent/CN112584853B/zh
Priority to EP18933038.4A priority patent/EP3845240A4/en
Priority to PCT/CN2018/105314 priority patent/WO2020051812A1/zh
Priority to US17/275,821 priority patent/US20210332100A1/en
Publication of WO2020051812A1 publication Critical patent/WO2020051812A1/zh

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to the technical field of polypeptide preparation methods, and in particular, to a novel structural design of proinsulin aspart and a method for preparing insulin aspart from the same.
  • 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, a total of 51 amino acids. A 7 (Cys) -B 7 (Cys) and A 20 (Cys) -B 19 (Cys) 4 cysteines form two disulfide bonds connecting the A chain and the B chain. In the A chain, A 6 (Cys) and A 11 (Cys) form intrachain disulfide bonds. Diabetes is characterized by elevated blood glucose levels due to lack of insulin secretion and / or increased hepatic glucose production. Insulin is a medicine for people with diabetes.
  • the overall goal in the development of insulin analogs is to mimic physiological insulin secretion, thereby improving glycemic control in patients with type 1 and type 2 diabetes (Berger M, A. Comment. Diabetes Res Clin Pract. 6: S25-S31, 1989. and Nosek L Et al., Diabetes, Obesity and Metabolism, 15: 77-83, 2013.).
  • Recent insulin analogs include additional amino acid residues or replacement of amino acid residues on natural insulin molecules by genetic engineering or biochemical reactions, or modifications to other functional groups. These modifications change the speed of biopharmaceutical efficiency by changing the pharmacological, pharmacokinetic and pharmacodynamic properties of insulin molecules, such as insulin glargine, insulin aspart, insulin lispro and so on.
  • Insulin aspart (U.S. patents US5618913, US5547930, US5834422) is a fast-acting insulin analog that rapidly separates and absorbs after subcutaneous injection, showing monomer stability. Insulin aspart, the natural insulin B 28 proline is replaced by aspartic acid, which enhances charge rejection, thereby further preventing hexamer formation. As a result, insulin aspart works more quickly and has a shorter duration of efficacy than normal human insulin. In addition, insulin aspart binds to plasma proteins to a lesser degree, and it is cleared from the blood faster than ordinary human insulin (Nosek L et al., Diabetes, Obesity and Metabolism, 15: 77-83, 2013; and Sanlioglu AD, etc. Human, Clinical utility of insulin and insulin analogs. Islets 5 (2): 67-78, 2013.).
  • proproinsulin In mammals, insulin is synthesized, processed, and stored in pancreatic ⁇ -cells.
  • the human insulin gene is located on the short arm of chromosome 11.
  • Insulin messenger RNA is translated into a 109 amino acid single-chain polypeptide precursor called proproinsulin.
  • Preproinsulin contains a 23 amino acid signal peptide.
  • the preproinsulin signal peptide is removed to form proinsulin.
  • Proinsulin consists of 3 parts: the amino-terminal B chain (30 amino acids), the hydroxyl-terminal A chain (23 amino acids), and an intermediate linking peptide called a C-peptide (35 amino acids).
  • proinsulin folds into its natural conformation, forming three disulfide bonds. Properly folded proinsulin is transported to the Golgi apparatus and packaged into secretory particles. In the Golgi apparatus, proinsulin undergoes proteolytic hydrolysis to produce active insulin consisting of two peptide chains, the A chain and the B chain. The A and B chains are connected by two disulfide bonds, and the C-peptide is released into a free peptide fragment. C-peptide restriction sites are located in two basic sequences (lysine 64, arginine, 65 and arginine 31, arginine 32).
  • Carboxypeptidase removes arginine 31 residues.
  • mature insulin is secreted into the blood circulation along with C-peptides to regulate blood sugar (Steiner DF, In. Pancreatic Beta Cell Health and Disease. Pp 31-49, 2008.).
  • the biological activity of proinsulin is only 10% of insulin.
  • Human insulin was the first proteinaceous drug produced by recombinant DNA technology. In 1978, human insulin was successfully expressed in the laboratory for the first time; in 1982, recombinant human insulin was approved as a therapeutic drug.
  • Recombinant human insulin precursor protein is biosynthesized by genetic modification and proteolytically cleaved to produce active insulin. Almost all publicly available insulin analogs are genetically engineered from human insulin genes and produced in E. coli or yeast.
  • the method for synthesizing recombinant human insulin was accomplished by the following method. For example, using the method of the E. coli expression system, either express a large fusion protein in the cytoplasm (Rich, DH et al., Pierce Chemical Company, Rockford. Pp.
  • one method expresses the A and B chains of insulin in E. coli, and then mixes the sulfonated A and B chains in vitro to form interchain disulfide bonds (Rich, DH et al. , Pierce Chemical Company, Rockford. Pp. 721-728, 1981; and Frank, BH et al. Pierce Chemical Company, Rockford. Pp. 729-738, 1981.).
  • this method also has disadvantages because it requires two separate fermentation processes and forms the correct disulfide bonds. Inappropriate disulfide bond formation between the sulfonated A and B chains results in reduced insulin yield. Overall, this method is very inefficient.
  • Another improved method uses a large fusion "ballast" (Chang, SG et al., Biochem. J. 329: 631-635, 1998; and Rhodes. CJ In: Diabetes Mellitus: A Fundamental and Clinical Text 3 rd ed.Pp 27, 2004.) to express proinsulin.
  • the method includes: cutting the fusion protein with cyanogen bromide to obtain proinsulin.
  • the proinsulin was sulfonated and separated, the refolded sulfonated proinsulin formed the correct in vitro disulfide bond, and then the C-peptide and product were cleaved with trypsin and carboxypeptidase B.
  • C-peptides in the folding of preproinsulin are not clear. There are two basic sites at both ends of the C-peptide, which are considered to be pro-insulin to insulin (Kroeff EP et al., J. Chromatogr. 461: 45-61, 1989; and Frank, Chance. Munch Med. Wschr., Suppl 1: S14-20, 1983.). In the production of recombinant insulin, there is a conserved, terminal bibasic amino acid sequence.
  • US patent US6875589 reports a novel miniproinsulin structure in which the C-peptide is shortened to one arginine residue.
  • This miniproinsulin can be converted to insulin by digestion with trypsin and carboxypeptidase.
  • This process did not produce Arg (A0) -insulin impurities.
  • this process uses cyanogen bromide to cleave the N-terminal peptide tag and then fold it again, which is inefficient, expensive and time consuming.
  • this patent is limited to the preparation of human insulin. There is no evidence that it can be used to make insulin analogs, especially insulin aspart.
  • the invention discloses a novel structural design of proinsulin aspart which can effectively improve the preparation process of recombinant insulin, and a method for preparing insulin aspart analogues.
  • the proinsulin structure provided by the present invention comprises a short C-peptide and a leader sequence, and is expressed by E. coli.
  • the invented polypeptide sequence is renatured with the leader peptide under correct renaturation conditions.
  • Insulin aspart is converted by trypsin and carboxypeptidase B in two steps.
  • the invention provides a novel genetic structure of proinsulin aspart for preparing insulin aspart analogs.
  • the proinsulin aspart sequence is shown in Formula I:
  • RR 1 is a leader peptide sequence that meets the following conditions:
  • R is a part of the superoxide dismutase (SOD) homologue, which is composed of 63 amino acids, including active methionine; 2 cysteine (C) residues are replaced by serine (S);
  • SOD superoxide dismutase
  • This leader peptide does not affect the renaturation of proinsulin aspart, and can be removed by cleavage;
  • cR 1 is either arginine or lysine.
  • R 2 is a C-peptide composed of either arginine or lysine
  • B 1 -B 27 represents the amino acid sequence of B 1 -B 27 in the B chain of natural human insulin
  • a 1 -A 21 represents the natural insulin A chain
  • B 28 is any one of aspartic acid, glutamic acid and proline, preferably aspartic acid;
  • B 29 is any one of lysine and proline, preferably lysine;
  • B 30 is any one of alanine and threonine, and is preferably threonine.
  • One aspect of the invention is a DNA sequence encoding proinsulin aspart described in Formula I.
  • the DNA sequence is optimized to ensure efficient expression of proinsulin aspart in a suitable host cell.
  • the content of the present invention includes ligating the above-mentioned new DNA sequence into a suitable vector, transferring the vector containing the new DNA sequence into E. coli, culturing E. coli introduced into a plasmid, and inducing new type of aspartogen in E. coli expression.
  • Another aspect of the invention is batch fed fermentation and production of insulin aspart, which includes the steps of culturing Escherichia coli expressing the formula I sequence proinsulin under suitable conditions; E. coli is treated with lysozyme and high pressure. Inclusion bodies containing proinsulin aspartate were obtained after incubation; the inclusion bodies were washed and purified to obtain relatively pure inclusion bodies, which were diluted and renatured after dissolution; renatured aspartins were trypsinized to remove the leading peptide to obtain the door.
  • Insulin-R 2 intermediate purified two-step ion exchange chromatography and one-phase reversed-phase preparative RP-HPLC chromatography to obtain purified aspart insulin-R 2 intermediate; after digestion with carboxypeptidase B, mature Insulin aspart; After purified by one-step reversed-phase RP-HPLC chromatography, purified aspart insulin is obtained; finally, the final insulin aspart drug is obtained by crystallization and drying.
  • the present invention relates to a sequence of a novel proinsulin aspart as preparation of an insulin aspart analog. Compared to the natural insulin, the amino acid at position B 28 of the B chain in the insulin aspart analog is improved to aspartic acid.
  • the novel pro-aspartic acid sequence is a single-chain polypeptide capable of being converted into an aspartic insulin analogue. It consists of a N-terminal leader peptide, an insulin A chain, a modified insulin B chain and an amino acid C-peptide.
  • the invention includes the addition of a leader peptide sequence to the N-terminus of the novel pro-aspartate.
  • An ideal leader peptide sequence should have the following characteristics:
  • the leader peptide sequence should not be too long, it will not cause additional metabolic burden on the host cells and adversely affect the fermentation process;
  • Lead peptide does not hinder the in vitro renaturation of the precursor, so that it can be normal renatured without removing the leader peptide, which protects the degradation of proinsulin aspart and enhances the solubility;
  • Lead peptide can increase the expression of fusion protein
  • the new proinsulin aspartyl described in the present invention can be converted to insulin aspart-R 2 by trypsin digestion (partial sequence of formula I).
  • the sequence composition of a novel proinsulin aspart as a precursor of insulin aspart provides an improved method for the preparation of recombinant insulin aspart analogs. Has fewer steps and fewer hazards in the process of promoting industrial safety and process management; eliminates time-consuming and costly purification steps, and improves finality by reducing false renaturation and incorrect enzymatic digestion of introduced process impurities Yield and purity of the product.
  • the present invention discloses a novel fusion protein structure for preparing insulin aspart analogues.
  • the proinsulin aspart sequence is shown in Formula I:
  • RR 1 is a leader peptide sequence that meets the following conditions:
  • R is a part of the superoxide dismutase (SOD) homologue, which is composed of 63 amino acids, including active methionine; 2 cysteine (C) residues are replaced by serine (S);
  • SOD superoxide dismutase
  • This leader peptide does not affect the refolding of proinsulin aspart, and can be removed by cleavage;
  • cR 1 is either arginine or lysine.
  • R 2 is a C-peptide composed of either arginine or lysine
  • B 1 -B 27 represents the amino acid sequence of B 1 -B 27 in the B chain of natural human insulin
  • a 1 -A 21 represents a natural human insulin A chain
  • B 28 is any one of aspartic acid, glutamic acid and proline, preferably aspartic acid;
  • B 29 is any one of lysine and proline, preferably lysine;
  • B 30 is any one of alanine and threonine, and is preferably threonine.
  • this R may be: MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP (SEQ ID NO: 1) or MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGP (SEQ ID)
  • SEQ ID NO: 3 arginine residue
  • SEQ ID NO: 4 a lysine residue
  • R 1 and R 2 may be the same amino acid, such as one of arginine or lysine.
  • Natural human insulin is composed of A chain and B chain.
  • the sequence of A chain is GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 5)
  • the sequence of B chain is FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 6).
  • the B chain in Formula I is modified from natural insulin.
  • the proline at position B 28 of natural insulin is replaced with aspartic acid, and B 27 , B 29 and B 30 are preferably natural amino acid residues.
  • the natural C-peptide sequence of human insulin is REALEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID NO: 7), and the C-peptide of the present invention is shortened to an arginine residue (SEQ ID ID NO: 8) or a lysine residue (SEQ ID ID NO: 9).
  • the molecular weight of proinsulin aspartide provided by the present invention is much smaller than that of human proinsulin.
  • sequence of natural human proinsulin is:
  • the artificial short B-chain insulin aspart precursor sequence is obtained by linking the A and B chains with three amino acid residues (AAK) in yeast.
  • the sequence is:
  • sequence of (B 1 -B 27 ) -B 28 -B 29 -B 30 -R 2- (A 1 -A 21 ) is one of SEQ ID NO 12 or SEQ ID NO 13;
  • sequence of SEQ ID NO 12 is:
  • sequence of SEQ ID NO 13 is:
  • the preferred sequence of proinsulin aspartide provided by the present invention includes any one of SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 or SEQ ID NO 17:
  • This proinsulin aspart may be used as a precursor for preparing insulin aspart analogs in E. coli or other host cells.
  • the new type of aspartins can be converted into insulin aspart by trypsin and carboxypeptidase B in two steps.
  • the sequence composition of novel proinsulin aspart provides an improved method for the production of recombinant insulin aspart analogs.
  • the genetic code for the new type of proinsulin is available as shown below.
  • mRNA or DNA a group of three adjacent nucleotides, also known as a triplet, encodes an amino acid called the genetic code.
  • An amino acid usually has one or more genetic codes called codon degeneration.
  • codon degeneration The following table shows the genetic code and corresponding amino acids.
  • Codon preference refers to the frequency of degradation in different organisms, even in the same species, with different coding genes. For efficient expression in a particular host, optimization of genetic codon selection and gene structure is required.
  • the present invention is embodied by providing an optimized gene sequence for the novel proinsulin aspart as shown in Formula I to ensure the effective expression of these proteins in E. coli.
  • Preferred gene sequences of the invention may include:
  • the invention includes ligating the optimized DNA sequence described above into a suitable expression vector and transferring it into a suitable host cell. Optimize appropriate fermentation conditions to achieve higher expression levels.
  • the expression vector mentioned in the present invention contains the above-mentioned nucleic acid sequence, is a vector that carries and expresses foreign genes into cells, and generally refers to a DNA plasmid.
  • the recombinant expression vector is preferably proinsulin aspart pET-API.
  • Expression plasmids must carry cis-acting components, such as promoter regions, transcription initiation sites, ribosome binding sites, and other DNA sequences. They usually carry antibiotic resistance genes for positive selection, such as the ⁇ -lactamase gene. (Ampicillin resistance), neomycin phosphotransferase (kanamycin resistance), and the like.
  • the expression plasmid carrying the target gene can be transferred into a suitable host cell by chemical or physical methods, and then positive clones can be selected by antibiotic resistance.
  • the expression host cell mentioned in the present invention refers to any cell capable of expressing a foreign gene, including mammalian cells, insect cells, yeasts, and various prokaryotic cells.
  • the preferred host cell is a prokaryotic cell, which may be any natural bacterium, such as E. coli, B. subtilis, or Salmonella, or it can be any improved variety that is more suitable for recombinant protein expression, such as E. coli DH5a, K12JM107, W3110, BL21 (DE3) , Rosetta and other strains.
  • Such host cells or microorganisms include the expression vectors described above.
  • the host cell or microorganism is preferably E. coli containing a recombinant expression vector of proinsulin aspart pET-API.
  • the method for preparing insulin aspart according to the present invention generally includes the following steps:
  • Aspartate-R 2 intermediate is digested with carboxypeptidase B and converted into mature insulin aspartate
  • FIG. 1 is a schematic diagram illustrating the preferred process steps for producing insulin aspart according to the implementation of the present invention.
  • An embodiment of the invention is the sequence SEQ ID NO: 14. In a preferred embodiment, the sequence is SEQ ID NO: 15. In another preferred embodiment, the sequence is SEQ ID NO: 16. In another preferred embodiment, the sequence is SEQ ID NO: 17.
  • the above-mentioned optimized genes are ligated into a suitable vector, such as a PTAC expression plasmid series, pGEX series or PET series, preferably a PET series plasmid, and more preferably a plasmid pET28a; the expression plasmid can be transfected with a K12JM109 engineering strain or K12W110 engineered bacteria to form expression clones.
  • the expression plasmid is transfected into a BL21 (DE3) engineered bacterium.
  • the expression clones can be grown to appropriate concentrations by shaking flasks or fermenters. Expression of pro-aspartate is then induced. Cells containing inclusion bodies expressing proinsulin aspart can be collected by centrifugation.
  • Insulin-containing cells were lysed by lysozyme treatment and high-pressure homogenization.
  • the isolated inclusion bodies are washed with a solution containing a detergent or a chaotropic agent at a low concentration and dissolved with a high pH buffer solution.
  • the lysis buffer contains Tris, EDTA, and L-cysteine.
  • the concentration of Tris is about 10-50 mM, the concentration of EDTA is about 0.05-1.00 mM, the concentration of L-cysteine is about 0.25-5.0 mM; preferably, the concentration of Tris is about 20-30 mM; the concentration of EDTA The concentration of L-cysteine is about 0.05-0.25 mM.
  • the pH of the solution When dissolved, the pH of the solution is about 11.6-12.4, preferably about 11.8-12.2; the temperature of the solution is about 10-30 ° C, preferably 15-25 ° C. Inclusion bodies dissolve for about 10-120 min, preferably 10-60 min. Insulin aspart will be refolded.
  • the pH of the solution is about 10.0-11.6, preferably about 10.8-11.4; the temperature of the solution is about 10-25 ° C, preferably about 15-20 ° C; the concentration of the total protein is about 1-10g / L, preferably about 1-7 g / L; renaturation duration is about 12-48h, preferably about 24-36h.
  • trypsin is added to the renatured asprosin solution
  • the pH value of trypsin digestion is about 8.0-10.0, preferably about 8.5-9.5
  • the concentration of trypsin is about 0.025-0.125 mg / g protein, preferably about 0.050-0.083 mg / g protein
  • the digestion temperature is about 15-37 ° C, preferably about 18-25 ° C
  • the digestion time is about 24-48h, preferably about 24-40h.
  • zinc ions with a final concentration of 3 mM were added to the trypsin digestion solution.
  • the pH of the solution was adjusted to 6.0. This resulted in flocculent precipitation of the aspart insulin-R 2 intermediate.
  • the insulin-aspartate-R 2 intermediate precipitate is dissolved with a suitable buffer, and purified by an appropriate method to obtain the insulin-aspartate-R 2 intermediate product.
  • the first step was purified by chromatography SP cation column insulin aspart -R 2 solution, to reach about 80% purity, followed by a second step SP cation column, to reach about 85% purity. Further purification can be performed using reverse phase preparation to remove impurities. The purity of the finally obtained insulin aspart-R 2 can reach more than 95%, and the content of the impurity DesB30-insulin is reduced to below 0.1%. In another better embodiment, the aspart insulin-R 2 solution is directly purified by preparative HPLC-RP. The purity of the final insulin aspart-R 2 can reach 95% or more.
  • a preparative HPLC-RP using an ammonium sulfate buffer system is used for the third column separation, wherein the concentration range of the ammonium sulfate is 0.1-0.3M, more preferably 0.15-0.2M; the pH value is 2.0-4.0 Within range; the organic modifier can be ethanol, methanol or acetonitrile. Insulin-R 2 was eluted with a linear concentration gradient of organic solvents.
  • a third column separation is performed using preparative HPLC-RP and a sodium sulfate buffer system, wherein the concentration range of sodium sulfate is 0.1-0.3M, more preferably 0.15-0.2M; the pH value is 2.0- Within the range of 4.0; the organic modifier may be ethanol, methanol or acetonitrile. Insulin-R 2 was eluted with a linear concentration gradient of organic solvents.
  • purification is performed using preparative HPLC-RP with a mixed sodium acetate and ammonium acetate buffer system, wherein the concentration of sodium acetate is in the range of 0.1-0.3M, more preferably 0.15-0.2M; The concentration range is 0.1-0.3M, more preferably 0.15-0.2M; the pH value is in the range of 2.0-4.0; the organic modifier may be ethanol, methanol or acetonitrile. Insulin-R 2 was eluted with a linear concentration gradient of organic solvents.
  • insulin aspart-R 2 was converted to insulin aspart by carboxypeptidase B.
  • the aspartate-R 2 intermediate was dissolved with a dissolution buffer.
  • the solution comprises Tris and EDTA.
  • the concentration of Tris is about 10-100 mM, and the concentration of EDTA is about 1-4 mM; more preferably, the concentration of Tris is about 20-30 mM, and the concentration of EDTA is about 2-4 mM.
  • the insulin-aspartate-R 2 intermediate was dissolved at pH 9.0. After lysis, carboxypeptidase B was added to the lysis solution.
  • the concentration of carboxypeptidase B is about 0.2-1.0 mg / g protein, preferably about 0.25-0.5 mg / g protein;
  • the pH value of carboxypeptidase B digestion is about 8.0-10.0, preferably about 8.0-9.0;
  • carboxypeptidase B digestion temperature is about 20-37 ° C, preferably about 20-30 ° C;
  • total protein concentration is 4-7g / L, preferably about 4-6g / L; carboxypeptidase B digestion The duration is about 3-24 h, preferably about 3-15 h.
  • the mature insulin aspart solution after carboxypeptidase B digestion was purified by preparative HPLC-RP to achieve a product purity of about 99%.
  • a preparative HPLC-RP using an ammonium sulfate buffer system is used for the fourth column separation, wherein the concentration range of the ammonium sulfate is 0.1-0.3M, more preferably 0.15-0.2M; the pH value is in the range of 2.0-4.0 Internal; the organic modifier can be ethanol, methanol or acetonitrile. Insulin aspart was eluted with a linear concentration gradient of organic solvents.
  • a fourth column separation is performed using preparative HPLC-RP and a sodium sulfate buffer system, wherein the concentration range of sodium sulfate is 0.1-0.3M, more preferably 0.15-0.2M; the pH value is 2.0- Within the range of 4.0; the organic modifier may be ethanol, methanol or acetonitrile. Insulin aspart was eluted with a linear concentration gradient of organic solvents.
  • the final purification is performed using a prepared HPLC-RP with a mixed sodium acetate, ammonium acetate and Tris buffer system, wherein the concentration of sodium acetate ranges from 0.05-0.3M, more preferably from 0.1-0.12M ;
  • concentration of ammonium acetate is 0.05-0.3M, more preferably 0.1-0.2M;
  • concentration range of the Tris buffer system is 0.05-0.3M, more preferably 0.075-0.2M;
  • the pH value is in the range of 7.1-8.5; organic modification
  • the sexual agent may be ethanol, methanol or acetonitrile. Insulin aspart was eluted with a linear concentration gradient of organic solvents.
  • insulin aspart eluting from HPLC-RP is precipitated with 3-7 mM zinc chloride and the precipitate is collected.
  • the collected precipitate was dissolved with a 5-20 mM hydrochloric acid solution, and the concentration of insulin aspart was adjusted to 3-15 mg / mL.
  • zinc acetate was added to the solution at a final concentration of 6 mM, and the mixture was stirred at room temperature for 2-3 hours, and then left at 10-20 ° C for 20-24 hours.
  • each gram of insulin aspart is washed with a 75% -100% ethanol solution, and the crystals are collected after washing; each gram of insulin aspart is washed again with 5-10 mL of absolute ethanol, and the crystals are collected after washing.
  • the crystals were transferred to a vacuum drying box and dried at 15-35 ° C for 60-96h, and the vacuum pressure was not greater than -0.08MPa.
  • the insulin aspart drug that is finally obtained in the preparation process described in the present invention exists in a crystal form.
  • the advantages of using the new proinsulin sequence as a precursor to produce aspart insulin are: In this process, E. coli fusion SOD homolog is used for expression, and a single amino acid is used as the C peptide, which effectively avoids Arg (A0) -insulin
  • the problem of impurities also makes the purification process easier, removing some time-consuming and expensive purification steps; because the C-peptide is an amino acid, reducing the quality loss of the enzyme conversion step; by reducing wrong refolding and wrong enzymes
  • the impurities caused by the cutting process improve the yield and purity of the final product.
  • FIG. 1 is a flowchart of a process for preparing insulin aspart using novel proinsulin aspart in E. coli according to the present invention.
  • Example 1 Construction of an E. coli clone expressing a new type of proinsulin aspart.
  • the N-terminal leader amino acid sequence can enhance expression and protect proinsulin aspart against degradation by E. coli.
  • the preferred leader amino acid sequence is
  • the C-terminus of this leader amino acid sequence is connected to the B chain of insulin aspart via an arginine or lysine residue, and the leader peptide is removed by trypsin cleavage.
  • the C-peptide of proinsulin aspart is shortened to an arginine or lysine residue.
  • the precursor sequence of the new proinsulin is:
  • Optimized gene sequence is: 5'ATGGCGACGCATGCCGTGAGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGCATGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTCATGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCTGGCTCTACCAGTGCAGGTCCGCGGTTTGTGAACCAGCATCTGTGCGGCAGCCATCTGGTGGAAGCGCTGTATCTGGTGTGCGGCGAACGCGGCTTCTTTTATACCGATAAAACCCGCGGCATTGTGGAACAGTGCTGCACCAGCATTTGCAGCCTGTATCAGCTGGAAAACTATTGCAACTAA 3 '(SEQ ID NO: 18).
  • the DNA fragment of SEQ ID NO 18 is chemically synthesized by a commercial CRO company.
  • a 5'NcoI site: CCATGG and a 3'Hind III site: AAGCTT are both contained in the synthetic DNA fragment.
  • the DNA fragment encoding the entire amino acid sequence of SEQ ID NO: 14 was cleaved with NcoI and Hind III restriction enzymes, and inserted into the pET 28a expression vector cleaved with the same restriction enzyme to form a pET-API expression vector.
  • the transformed pET-API expression vector was transfected into BL21 (DE3) E. coli. Positive clones were screened by kanamycin resistance and confirmed by DNA sequencing. Positive clones were cultured and expanded, and then sterile media and glycerol were added to the cells. 1 mL of the cell culture solution was transferred into a sterile ampule and stored at -80 ° C to form a proinsulin aspart working seed bank (WCB).
  • BL21 DE3 E. coli
  • Positive clones were screened by kanamycin resistance and confirmed by DNA sequencing. Positive clones were cultured and expanded, and then sterile media and glycerol were added to the cells. 1 mL of the cell culture solution was transferred into a sterile ampule and stored at -80 ° C to form a proinsulin aspart working seed bank (WCB).
  • WBC proinsulin aspart working seed bank
  • Example 2 Expression of proinsulin asp fusion protein
  • the WCB obtained in Example 1 was cultured in LB medium (containing 1.5% w / v yeast powder and 0.5% w / v sodium chloride) at 37 ° C for 8 hours to obtain a cell seed solution.
  • the seed solution was inoculated into BFM medium (containing 0.6% w / v diammonium phosphate, 0.4% w / v ammonium chloride, 1.35% w / w potassium dihydrogen phosphate, 0.139% w / w magnesium sulfate 7 water, 0.28 % W / w citric acid monohydrate, 0.8% w / w glucose, 0.3% w / w yeast powder and 1% mL / L trace element solution), further cultured for 8 hours to obtain a secondary seed solution, and then inoculated at a volume ratio of 1:10 To the fermentation tank.
  • the cells of the inclusion body containing proinsulin aspart were resuspended in a buffer solution of 25 mM Tris, 10 mM EDTA, pH 8.0 to control the cell concentration to 200 g / L.
  • the bacterial cells were lysed by lysozyme treatment and high-pressure homogenization. The bacterial lysate was centrifuged to collect the inclusion body pellets and remove the supernatant.
  • inclusion bodies Wash the inclusion bodies with a washing solution of 25 mM Tris, 1 M urea, 1% Tween 20, pH 8.0. After washing, the inclusion bodies were resuspended with 25 mM Tris, 0.1 mM EDTA, and 0.5 mM L-cysteine buffer, the pH was adjusted to 12, and dissolved at 15-25 ° C for 10-60 min. The dissolved solution was named as inclusion body lysate.
  • the inclusion body solution 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 renaturation was performed for 24-48 h to obtain renatured aspartogen.
  • the B31Arg-aspartic acid precipitation was dissolved in a buffer solution of 3% isopropanol and 50 mM acetic acid, and the dissolution pH was 3.5.
  • the dissolved B31Arg-aspartic insulin was loaded as a sample on an SP chromatography column and equilibrated with the same buffer as the dissolution buffer.
  • B31Arg-aspartic acid can be eluted with a 30% isopropanol, 1M sodium chloride gradient.
  • the purity of B31Arg-aspartic acid after purification by 2 SP chromatography was about 85%.
  • B31Arg-aspartic acid eluate was added with 5 mM zinc chloride, and the pH was adjusted to 6.5 to form a flocculent precipitate of B31Arg-aspartic acid.
  • B31Arg-aspartic insulin was above 98%, and the content of DesB30-aspartic insulin was reduced to below 0.1%.
  • a high-purity B31Arg-aspartic acid intermediate was obtained.
  • the purified B31Arg-aspartic acid intermediate precipitate was resuspended in 25 mM Tris, 2 mM EDTA, and adjusted to pH 9.0 for dissolution. After the dissolution was completed, the pH of the solution was adjusted to 8.5, and a final concentration of 0.33 mg / g protein was added to the dissolving solution. Digestion was performed at 25 ° C for 3 h, and the C-peptide arginine residue was removed to obtain insulin aspart. This process can be detected by HPLC-RP (C18). After the digestion is completed, the pH value of the digestion solution is adjusted to 3.8, and the digestion is terminated.
  • the transformed insulin aspart was loaded onto a reversed-phase preparative chromatography column.
  • the column was equilibrated with a solution obtained by mixing a solution of 0.1M ammonium acetate, 0.1M sodium acetate, 0.1M Tris, and pH 7.5 with acetonitrile at a ratio of 9: 1.
  • the elution buffer is a solution obtained by mixing a solution of 0.1M ammonium acetate, 0.1M sodium acetate, 0.1M Tris, and pH 3.5 with acetonitrile according to 1: 9. Insulin aspart was eluted with a linear gradient of elution buffer.
  • the crystals were collected by suction filtration. The crystals were then washed with 5 ml of a 75% ethanol solution / (g insulin aspart), and the crystals were collected after washing. The crystals were then washed again with 10 ml of an ethanol solution / (g of aspart insulin), and the crystals were collected after the washing was completed. The crystals were transferred to a vacuum drying box and dried at a temperature of 25 ° C. for 80 h. The vacuum pressure was not greater than -0.08 MPa. After drying, the final insulin aspart drug was obtained.

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Abstract

本发明提供了一种新型门冬胰岛素原的结构设计和门冬胰岛素制备方法。其中主要步骤包括设计门冬胰岛素原的序列，构建重组门冬胰岛素工程菌，通过工程菌诱导以包涵体形式表达门冬胰岛素融合蛋白，再经过变性、复性、酶切和分离纯化得到成熟的门冬胰岛素原料药。发明通过改变重组前导肽和C-肽序列，避免使用溴化氰切割这种危险而繁琐的步骤；缩短C-肽为1个氨基酸，减少酶切转化的质量损失。

Description

一种新型门冬胰岛素原的结构和制备门冬胰岛素的方法 技术领域
本发明涉及多肽制备方法技术领域，具体涉及一种新型的短门冬胰岛素原的结构设计和由其制备门冬胰岛素的方法。
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)形成链内二硫键。糖尿病的特点是由于胰岛素分泌缺乏和/或增加的肝葡萄糖生产而导致的血糖水平升高。胰岛素是针对糖尿病患者的药物。
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.和Nosek L等人,Diabetes,Obesity and Metabolism,15:77-83,2013.)。
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，如甘精胰岛素、门冬胰岛素、赖脯胰岛素等。
门冬胰岛素(美国专利US5618913，US5547930，US5834422)是一种快速作用的胰岛素类似物，在皮下注射后迅速分离和吸收，显示了单体的稳定性。门冬胰岛素，天然胰岛素的B 28脯氨酸被天冬氨酸代替，增强电荷排斥，从而进一步阻止六聚体形成。因此，与正常的人胰岛素相比，门冬胰岛素的作用更迅速，效力持续时间更短。此外，门冬胰岛素与血浆蛋白的结合程度较低，它比普通人胰岛素更快的从血液中清除(Nosek L等人,Diabetes,Obesity and Metabolism,15:77-83,2013；和Sanlioglu AD等人,Clinical utility of insulin and insulin analogs.Islets 5(2):67-78,2013.)。
在哺乳动物中，胰岛素是在胰腺β-细胞内进行合成、加工和储存的。人胰岛素基因位于11号染色体的短臂上。胰岛素的信使RNA被翻译成109个氨基酸的单链多肽前体，称为前胰岛素原。前胰岛素原含有一种23个氨基酸的信号肽。在转移到粗糙型内质网期间，将前胰岛素原的信号肽去除，形成胰岛素原。胰岛素原由3个部分组成：氨基末端B链(30个氨基酸)， 羟基末端A链(23个氨基酸)和被称为C-肽(35个氨基酸)的中间连接肽。在内质网内，胰岛素原折叠成天然的构象，形成3个二硫键。正确折叠的胰岛素原被转运到高尔基体并被包装成分泌颗粒，在高尔基体内胰岛素原经历了蛋白酶水解，产生由A链和B链2条肽链组成的有活性的胰岛素。A链和B链，由2个二硫键连接，同时释放C-肽成为一个自由的肽片段。C-肽有限制性的酶切位点位于2个基本序列(赖氨酸64，精氨酸，65和精氨酸31，精氨酸32)，在C-肽被酶切去除后，通过羧肽酶去除精氨酸31残基。当细胞受到刺激时，成熟的胰岛素，连同C-肽一起被分泌到血液循环中，以调节血糖(Steiner DF,In.Pancreatic Beta Cell in Health and Disease.Pp 31-49,2008.)。胰岛素原的生物活性仅为胰岛素的10％。
人胰岛素是重组DNA技术生产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是由遗传修饰的生物合成，并通过蛋白水解切割生成有活性的胰岛素。几乎所有公开出售的胰岛素类似物都是用基因工程技术从人胰岛素基因中改良出来的，并在大肠杆菌或酵母中生产。重组人胰岛素的合成方法是用以下方法完成的。例如，使用大肠杆菌表达***的方法，要么在细胞质中表达一个大的融合蛋白(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.)，要么使用一个信号肽，使分泌物进入到胞质空间(Chan,S.J.等人，Proc.Natl.Acad.Sci.USA.78(9):5401-5404,1981.)中。同时也报道了一种利用酵母，尤其是酿酒酵母，来表达和分泌胰岛素前体的方法(Thim,L.等人，Proc.Natl.Acad.Sci.USA.83:6766-6770,1986.)。
对于使用转基因大肠杆菌的方法，一种方法在大肠杆菌中分别表达了胰岛素的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，这种方法也有缺点，因为它需要两个独立的发酵过程，并形成正确的二硫键。磺化A链和B链之间不恰当的二硫键形成导致胰岛素的产率降低。总的来说，这种方式生产效率很低。
据报道，另一种改进的方法，用一个大的融合―压舱物‖(Chang,S.G.等人，Biochem.J.329:631-635,1998；和Rhodes.C.J.In:Diabetes Mellitus:A Fundamental and Clinical Text 3 rd ed.Pp 27,2004.)来表达胰岛素原。方法包括：用溴化氰切断融合蛋白，获得胰岛素原。对胰岛素原进行磺化和分离，重折叠磺化胰岛素原形成正确的体外二硫键，然后用胰蛋白酶和羧肽酶B切开C-肽和产物。为了生成胰岛素，聚变―压舱物‖肽的巨大尺寸限制了产量，并可能阻碍重新折叠的过程——在重新折叠之前，需要将融合―压舱物‖去除。采用溴化氰切割大融合蛋 白形成了一种工作环境危害，需要高水平的劳动保护。因此，上述工艺需要承担在制备过程中使用繁琐的纯化步骤所造成的不便。
另一种方法是由诺和诺德公司开发的(欧洲专利EP0163529，美国专利US4916212)，它包含了由缩短的B链(desThr30)和通过酵母中的三个氨基酸残基(AAK)连接的A链，并通过发酵、纯化、酶切反应、酸性水解和多步纯化等步骤分离出胰岛素。然而，该过程导致胰岛素的产率低得令人无法接受。事实上，酵母所提供的胰岛素产量相对较低。与大肠杆菌相比，酵母表达***中固有的表达水平较低，发酵周期较长。
C-肽在前胰岛素原的折叠中所起的作用还不是很清楚。在C-肽的两端都是两个碱性的位点，被认为是促胰岛素原转化为胰岛素(Kroeff EP等人，J.Chromatogr.461:45-61,1989；和Frank,Chance.Munch Med.Wschr.,Suppl 1:S14-20,1983.)的必要条件。在重组胰岛素生产中，有一个保守的、末端双碱性氨基酸序列，据报道，通过胰蛋白酶酶切胰岛素原时有错误酶切的杂质产生，导致杂质存在酶切混合物中(例如，Arg(A0)-胰岛素)(Wetzel,R.等人，Gene.16:63-71,1981.)。为了去除这些工艺杂质，需要额外的纯化步骤，这可能会导致额外的产量损失，并使过程耗费时间和成本。
有几项实验(EP0055945，EP0163529，US6875589)(Dixon M,Webb EC Enzymes 3 rd ed,London:Longman Group Ltd.,pp.261-262，1979.)报道，在体外进行胰岛素复性，完整的C-肽是不必要的。一种观点认为，C-肽的功能是连接A链和B链，促进了前胰岛素原的复性，形成了正确的二硫键。Change Et Al.(1998)报道说，在复性过程中，短序列比长序列的C-肽更有效。例如，美国专利US6875589报道了一种新型的微型胰岛素原结构，其中的C-肽被缩短为一个精氨酸残基。这种微型胰岛素原可以通过胰蛋白酶和羧肽酶酶切来转化成胰岛素。这个过程没有产生Arg(A0)-胰岛素杂质。然而，这个过程利用了溴化氰来切割N端的肽标签，然后再折叠，这是低效、昂贵和耗时的。此外，这项专利仅限于用于制备人胰岛素。没有证据表明它可以用来制备胰岛素类似物，特别是门冬胰岛素。
综上所述，现在急需一种更高效和更完善的门冬胰岛素生产工艺，这种门冬胰岛素制备工艺具有产生较低的工艺杂质和较高的纯化效果。
发明内容：
本发明公开一种能够有效改善重组胰岛素的制备工艺的新型的短门冬胰岛素原的结构设计，以及制备门冬胰岛素类似物的方法。
为了实现上述目的，本发明提供的短门冬胰岛素原结构包含一种短的C-肽和一个前导序列，是用大肠杆菌来表达的。所发明的多肽序列是带着前导肽一起在正确的复性条件下进行复性。门冬胰岛素是经过胰蛋白酶和羧肽酶B两步酶切转化而成的。
溴化氰切割这种危险而繁琐的步骤在这个新工艺中被排除在外，因为新发明的门冬胰岛素原在有前导肽存在情况下，可以有效地折叠成其天然结构。
本发明提供了一种新型的短门冬胰岛素原的基因结构用于制备门冬胰岛素类似物。门冬胰岛素原序列如公式I所示：
R-R 1-(B 1-B 27)-B 28-B 29-B 30-R 2-(A 1-A 21)
其中：
R-R 1是前导肽序列，满足以下条件：
a.R是超氧化物歧化酶(SOD)同系物的一部分，它由63个氨基酸组成，包括活性甲硫氨酸；2个半胱氨酸(C)残基由丝氨酸(S)取代；
b.这个前导肽不会影响门冬胰岛素原的复性，并且能够裂解去除；
c.R 1是精氨酸和赖氨酸中的任意一个。
R 2是由精氨酸或者赖氨酸中的任意一个构成的C-肽；
B 1-B 27表示天然人胰岛素B链中B 1-B 27的氨基酸序列
A 1-A 21表示天然胰岛素A链；
B 28是天冬氨酸，谷氨酸和脯氨酸中的任意一个，优选为天冬氨酸；
B 29是赖氨酸和脯氨酸中的任意一个，优选为赖氨酸；
B 30是丙氨酸和苏氨酸中的任意一个，优选为苏氨酸。
本发明一方面是编码公式I描述的门冬胰岛素原的DNA序列。DNA序列经过优化，以确保在合适的宿主细胞内有效的表达门冬胰岛素原。
本发明内容包括将上述的新的DNA序列连接到合适的载体中，将含有新的DNA序列的载体转入大肠杆菌内，培养导入质粒的大肠杆菌和诱导新型门冬胰岛素原在大肠杆菌中进行表达。
本发明的另一方面是门冬胰岛素的分批补料发酵和生产，包含的步骤有在合适的条件下培养大肠杆菌表达公式I序列的门冬胰岛素原；大肠杆菌经溶菌酶处理和高压均质后获得含有门冬胰岛素原的包涵体；包涵体经过洗涤纯化后得到较纯的包涵体，经过溶解后进行稀释复性；复性的门冬胰岛素原通过胰蛋白酶酶切去除前导肽得到门冬胰岛素-R 2中间体；采用两步离子交换色谱法和一步反相制备RP-HPLC色谱法制备获得精制的门冬胰岛素-R 2中间体； 经过羧肽酶B酶切后，形成成熟的门冬胰岛素；再经过一步反相RP-HPLC色谱法纯化后，得到精制的门冬胰岛素；最后通过结晶和干燥，得到最终门冬胰岛素原料药。
本发明涉及一种制备门冬胰岛素类似物的新型门冬胰岛素原的序列。与天然的胰岛素相比，门冬胰岛素类似物中B链的B 28位氨基酸改良为天冬氨酸。
新型的门冬胰岛素原序列是指能够转化为门冬胰岛素类似物的单链多肽，由N端的前导肽，胰岛素A链，改良后的胰岛素B链和一个氨基酸的C-肽组成。
在大肠杆菌表达中，短C-肽和小分子量的负效应使表达的融合蛋白易于降解。为了避免这种效应，本发明包括在新型的门冬胰岛素原的N端加入前导肽序列。理想的前导肽序列应该具有以下特征：
1.前导肽序列不宜过长，不会对宿主细胞产生额外的代谢负担和对发酵过程产生不利影响；
2.前导肽不会阻碍前体的体外复性，使得在不去除前导肽的情况下可以正常复性，这保护了短门冬胰岛素原的降解和增强了溶解度；
3.前导肽能够提高融合蛋白的表达量；
4.前导肽很容易去除。
在本发明中阐述的新型门冬胰岛素原能够通过胰蛋白酶酶切转换成门冬胰岛素-R 2(公式I的部分序列)。作为制备门冬胰岛素前体的新型门冬胰岛素原的序列组成，为重组门冬胰岛素类似物的制备提供了一个改良的方法。在促进工业安全和工艺管理的过程中具有较少的步骤和较少的危害；消除了耗时和昂贵的纯化步骤，并通过减少错误的复性和错误的酶切引入的工艺杂质来提高最终产品的收率和纯度。
因此，本发明公开了一种新的用于制备门冬胰岛素类似物的融合蛋白结构。门冬胰岛素原序列如公式I所示：
R-R 1-(B 1-B 27)-B 28-B 29-B 30-R 2-(A 1-A 21)
其中，
R-R 1是前导肽序列，满足以下条件：
a.R是超氧化物歧化酶(SOD)同系物的一部分，它由63个氨基酸组成，包括活性甲硫氨酸；2个半胱氨酸(C)残基由丝氨酸(S)取代；
b.这个前导肽不会影响门冬胰岛素原的重折叠，并且能够裂解去除；
c.R 1是精氨酸和赖氨酸中的任意一个。
R 2是由精氨酸或者赖氨酸中的任意一个构成的C-肽；
B 1-B 27表示天然人胰岛素B链中B 1-B 27的氨基酸序列；
A 1-A 21表示天然人胰岛素A链；
B 28是天冬氨酸，谷氨酸和脯氨酸中的任意一个，优选为天冬氨酸；
B 29是赖氨酸和脯氨酸中的任意一个，优选为赖氨酸；
B 30是丙氨酸和苏氨酸中的任意一个，优选为苏氨酸。
具体实施例中，这个R可能是：MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP(SEQ ID NO:1)或者是：MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGP(SEQ ID NO:2)。前导肽序列的C端通过一个精氨酸残基(SEQ ID NO:3)或者赖氨酸残基(SEQ ID NO:4)与短门冬胰岛素原相连接。
具体实施例中，其中R 1和R 2可能是相同的氨基酸，如精氨酸或赖氨酸中的一种。
天然人胰岛素由A链和B链构成，其中A链序列为GIVEQCCTSICSLYQLENYCN(SEQ ID NO:5)，B链序列为FVNQHLCGSHLVEALYLVCGERGFFYTPKT(SEQ ID NO:6)。根据这项发明，公式I中的B链由天然胰岛素改良得到的。对于门冬胰岛素原，天然胰岛素的B 28位脯氨酸被天冬氨酸所取代，B 27、B 29和B 30最好是天然的氨基酸残基。
人胰岛素的天然C-肽序列是RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR(SEQ ID NO:7)，本发明的C-肽缩短成一个精氨酸残基(SEQ ID NO:8)或者赖氨酸残基(SEQ ID NO:9)。本发明提供的门冬胰岛素原的分子量比人胰岛素原的分子量小很多。
天然人胰岛素原的序列为：
Figure PCTCN2018105314-appb-000001
另外人工短B链的门冬胰岛素前体序列是通过在酵母中用三种氨基酸残基(AAK)连接A链和B链，序列为：
Figure PCTCN2018105314-appb-000002
具体实施例中(B 1-B 27)-B 28-B 29-B 30-R 2-(A 1-A 21)的序列是SEQ ID NO 12或SEQ ID NO 13中的一种；
其中SEQ ID NO 12的序列为：
Figure PCTCN2018105314-appb-000003
SEQ ID NO 13的序列为：
Figure PCTCN2018105314-appb-000004
本发明提供的优选的门冬胰岛素原的序列包括SEQ ID NO 14、SEQ ID NO 15、SEQ ID NO 16或SEQ ID NO 17中的任意一种：
Figure PCTCN2018105314-appb-000005
这种门冬胰岛素原可作为在大肠杆菌或其它宿主细胞中制备门冬胰岛素类似物的前体。新型的门冬胰岛素原可以通过胰蛋白酶和羧肽酶B经过两步酶切转化成门冬胰岛素。新型门冬胰岛素原的序列组成，为重组门冬胰岛素类似物的生产提供了一个改良的方法。
可以获得新型门冬胰岛素原的基因编码，如下所示。在mRNA或DNA中，一组三种相邻的核苷酸，也称为三元组，它编码一种氨基酸，称为遗传密码。一种氨基酸通常有一个或多个遗传密码，称为密码子简并。下表显示了遗传密码和相应的氨基酸。
Figure PCTCN2018105314-appb-000006
密码子偏好性指的是不同生物体中退化的使用频率，即使是在相同的物种中，不同的编码基因也是如此。为了在特殊的宿主中有效表达，需要对遗传密码子选择和基因结构进行优化。本发明体现在包括为如公式I所示的新型门冬胰岛素原提供优化的基因序列，以确保这些蛋白质在大肠杆菌中的有效表达。本发明的首选基因序列可能包括：
Figure PCTCN2018105314-appb-000007
Figure PCTCN2018105314-appb-000008
本发明包括将上述优化的DNA序列连接到合适的表达载体中，并将其转入至合适的宿主细胞。优化适当的发酵条件，以达到较高的表达水平。
本发明中提到的表达载体包含上述的核酸序列，是携带和表达外来基因进入细胞的载体，通常指的是DNA质粒。所述的重组表达载体优选为门冬胰岛素原pET-API。表达质粒必须携带顺式作用组件，如启动子区域、转录起始位点、核糖体结合位点和其他的DNA序列，通常携带抗生素耐药性基因用于阳性选择，如β-内酰胺酶基因(氨苄青霉素抗性)、新霉素磷酸转移酶(卡那霉素抗性)等。携带目标基因的表达质粒可以通过化学或物理的方法转入到合适的宿主细胞，然后通过抗生素耐药性来选择阳性的克隆体。
本发明中提到的表达宿主细胞指的是任何能够表达外源基因的细胞，包括哺乳动物细胞、昆虫细胞、酵母菌和各种原核细胞。首选的宿主细胞是原核细胞，可能是任何天然细菌，如大肠杆菌、枯草杆菌、沙门氏菌，或者它可以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和其它菌株。这种宿主细胞或微生物包含上述的表达载体。所述宿主细胞或微生物优选为包含门冬胰岛素原pET-API重组表达载体的大肠杆菌。
根据具体情况，本发明所述的制备门冬胰岛素的方法通常包括以下步骤：
1.根据公式I的序列设计和构建门冬胰岛素原的序列；
2.发酵和表达门冬胰岛素原；
3.包涵体释放和溶解，以及门冬胰岛素原的复性；
4.门冬胰岛素原被胰蛋白酶酶切转化成门冬胰岛素-R 2中间体；
5.门冬胰岛素-R 2中间体的纯化；
6.门冬胰岛素-R 2中间体被羧肽酶B酶切转化为成熟的门冬胰岛素；
7.经结晶和干燥后得到门冬胰岛素原料药。图1是根据本发明的实施情况，阐述生产门冬胰岛素的优选工艺步骤的示意图。
本发明的一个实施例是序列SEQ ID NO：14。在一个优选实施例中，序列是SEQ ID NO:15。在另一个优选实施例中，序列是SEQ ID NO:16。在另一个优选实施例中，序列是SEQ ID NO:17。
在一个实施方案中，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；该表达质粒可转染K12JM109工程菌或K12W110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。该表达克隆可通过摇瓶或发酵罐培养至适当浓度。然后诱导门冬胰岛素原的表达。含有表达门冬胰岛素原的包涵体的细胞可以通过离心收集。
将含有门冬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。在一个实施方案中，溶解缓冲液含有Tris、EDTA和L-半胱氨酸。Tris的浓度约为10-50mM，EDTA的浓度约为0.05-1.00mM，L-半胱氨酸的浓度约为0.25-5.0mM；优选的是，Tris的浓度约为20-30mM；EDTA的浓度约为0.05-0.25mM，L-半胱氨酸的浓度约为0.25-1.0mM。当溶解时，溶液的pH值约为11.6-12.4，优选为约11.8-12.2；溶液的温度约为10-30℃，优选为15-25℃。包涵体溶解约10-120min，优选10-60min。门冬胰岛素原将被重新折叠。在一个实施方案中，溶液的pH值为约10.0-11.6，优选为约10.8-11.4；溶液的温度约为10-25℃，优选约15-20℃；总蛋白的浓度为约1-10g/L，优选约1-7g/L；复性持续时间约为12-48h，优选为约24-36h。
在一个实施方案中，胰蛋白酶被添加到复性完成的门冬胰岛素原溶液中，胰蛋白酶消化的pH值约为8.0-10.0，优选约8.5-9.5；胰蛋白酶的浓度约为0.025-0.125mg/g蛋白质，优选约0.050-0.083mg/g蛋白质；酶切温度约15-37℃，优选为约18-25℃，酶切时间约24-48h，优选为约24-40h。酶切完成后，将最终浓度为3mM的锌离子加入到胰蛋白酶酶切液中。溶 液pH值调节到6.0。这使得门冬胰岛素-R 2中间体形成絮状沉淀。用合适的缓冲液对门冬胰岛素-R 2中间体沉淀物进行溶解，并用适当的方法纯化得到门冬胰岛素-R 2中间体产物。
在一个实施方案中，通过第一步SP阳离子柱色谱纯化门冬胰岛素-R 2溶液，以达到约80％的产品纯度，接着是第二步SP阳离子柱，以达到约85％的产品纯度。进一步的纯化可以用反相制备进行，以除去杂质。最终得到的门冬胰岛素-R 2纯度可达95％以上，杂质DesB30-门冬胰岛素含量降至0.1％以下。在另一个更好的实施方案中，门冬胰岛素-R 2溶液通过制备性HPLC-RP直接纯化。最终的门冬胰岛素-R 2的纯度可达到95％或更多。在一个实施方案中，使用硫酸铵缓冲体系的制备HPLC-RP进行第三次柱分离，其中硫酸铵的浓度范围为0.1-0.3M，更优选为0.15-0.2M；pH值在2.0-4.0的范围内；有机改性剂可以是乙醇、甲醇或乙腈。用有机溶剂的线性浓度梯度洗脱门冬胰岛素-R 2。在另一个优选实施方案中，使用制备HPLC-RP与硫酸钠缓冲体系进行第三次柱分离，其中硫酸钠的浓度范围为0.1-0.3M，更优选为0.15-0.2M；pH值在2.0-4.0的范围内；有机改性剂可以是乙醇、甲醇或乙腈。用有机溶剂的线性浓度梯度洗脱门冬胰岛素-R 2。在另一个更优选的实施方案中，使用制备HPLC-RP与混合乙酸钠和乙酸铵缓冲体系进行纯化，其中乙酸钠的浓度范围为0.1-0.3M，更优选为0.15-0.2M；乙酸铵的浓度范围为0.1-0.3M，更优选0.15-0.2M；pH值在2.0-4.0范围内；有机改性剂可以是乙醇、甲醇或乙腈。用线性浓度梯度的有机溶剂洗脱门冬胰岛素-R 2。
第三次柱分离后，门冬胰岛素-R 2经羧肽酶B转化为门冬胰岛素。首先，用溶解缓冲液溶解门冬胰岛素-R 2中间体。在一个实施方案中，所述溶液包含Tris和EDTA。Tris的浓度约为10-100mM，EDTA的浓度约为1-4mM；更优选的是，Tris的浓度约为20-30mM，EDTA的浓度约为2-4mM。门冬胰岛素-R 2中间体在pH为9.0时溶解。溶解后，将羧肽酶B加入到溶解溶液中。在一个实施方案中，羧肽酶B的浓度约为0.2-1.0mg/g蛋白质，优选约0.25-0.5mg/g蛋白质；羧肽酶B酶切的pH值约为8.0-10.0，优选约为8.0-9.0；羧肽酶B酶切温度约为20-37℃，优选约20-30℃；总蛋白浓度为4-7g/L，优选约4-6g/L；羧肽酶B酶切的持续时间约3-24h，优选约3-15h。
羧肽酶B酶切后的成熟门冬胰岛素溶液通过制备HPLC-RP纯化，以达到约99％的产品纯度。在一个实施方案中，使用硫酸铵缓冲体系的制备HPLC-RP进行第四柱分离，其中硫酸铵的浓度范围为0.1-0.3M，更优选为0.15-0.2M；pH值在2.0-4.0的范围内；有机改性剂可以是乙醇、甲醇或乙腈。用有机溶剂的线性浓度梯度洗脱门冬胰岛素。在另一个优选实施方案中，使用制备HPLC-RP与硫酸钠缓冲体系进行第四次柱分离，其中硫酸钠的浓度范围为0.1-0.3M，更优选为0.15-0.2M；pH值在2.0-4.0的范围内；有机改性剂可以是乙醇、甲醇或 乙腈。用有机溶剂的线性浓度梯度洗脱门冬胰岛素。在另一个优选的实施方案中，使用制备的HPLC-RP与混合的乙酸钠、乙酸铵和Tris缓冲体系进行最终纯化，其中乙酸钠的浓度范围为0.05-0.3M，更优选为0.1-0.12M；乙酸铵的浓度为0.05-0.3M，更优选为0.1-0.2M；Tris缓冲体系的浓度范围为0.05-0.3M，更优选为0.075-0.2M；pH值在7.1-8.5范围内；有机改性剂可为乙醇、甲醇或乙腈。用有机溶剂的线性浓度梯度洗脱门冬胰岛素。
在一个实施方案中，从HPLC-RP洗脱的门冬胰岛素用3-7mM的氯化锌进行沉淀，收集沉淀。收集到的沉淀用5-20mM的盐酸溶液进行溶解，并且将门冬胰岛素的浓度调节为3-15mg/mL。往门冬胰岛素溶解液中加入终浓度为50-200mM的氯化钠，20-100m柠檬酸三钠二水合物，0.2％-1.0％(v/v)间甲酚和10-30％(v/v)无水乙醇，调节pH6.0-6.3。最终，往溶液中加入终浓度为6mM的乙酸锌后室温搅拌2-3h，然后在10-20℃放置20-24h。结晶完成后，每克门冬胰岛素用75％-100％的乙醇溶液进行洗涤，洗涤后收集晶体；每克门冬胰岛素用5-10mL的无水乙醇进行再次洗涤，洗涤后收集晶体。将晶体转移到真空干燥箱中，在15-35℃下干燥60-96h，真空压力不大于-0.08MPa。
本发明描述的制备工艺最终得到的门冬胰岛素原料药是以晶体形式存在的。
使用新型门冬胰岛素原序列作为前体生产门冬胰岛素生产工艺的优势是：在这个工艺中采用大肠杆菌融合SOD同系物进行表达，采用单个氨基酸作为C肽，有效避免了Arg(A0)-胰岛素杂质问题，也使得纯化工艺变得更加的简便，去除了一些费时和昂贵的纯化步骤；因为C-肽就一个氨基酸，减少酶转化步骤的质量损失；；通过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；相对现有技术中的酵母表达体系，本专利申请中采用大肠杆菌的包涵体表达和碱溶解复性，可获得较高的收率，且发酵周期大大缩短；开发了胰蛋白酶消化促进门冬胰岛素分子的成熟，避免使用剧毒危险试剂溴化氰，提高了工艺的可操作性，提供了安全的工作环境。因此，制备高质量门冬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，在大肠杆菌中使用新型门冬胰岛素原制备门冬胰岛素的工艺流程图。
具体实施方式
为便于本领域技术人员理解本发明内容，下面将结合具体实施例进一步描述本发明的技术方案，但以下内容不应以任何方式限制本发明权利要求书请求保护的范围。
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。
实例1：构建表达公式Ⅰ新型门冬胰岛素原的大肠杆菌克隆。
设计一个用于在大肠杆菌中表达如公式I所示的门冬胰岛素原蛋白序列。N端先导氨基酸序列可以增强表达，保护门冬胰岛素原，防止被大肠杆菌降解。首选的前导氨基酸序列是
Figure PCTCN2018105314-appb-000009
该前导氨基酸序列的C端通过精氨酸或赖氨酸残基连接到门冬胰岛素的B链，前导肽通过胰蛋白酶裂解被除去。门冬胰岛素原的C-肽被缩短为一个精氨酸残基或赖氨酸残基，新型门冬胰岛素原的前体序列为：
Figure PCTCN2018105314-appb-000010
门冬胰岛素原带前导肽的全序列MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN(SEQ ID NO:14)。为了确保在大肠杆菌中有效表达SEQ ID 14的蛋白质，遗传密码进行了优化。优化的基因序列是：5’ATGGCGACGCATGCCGTGAGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGCATGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTCATGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCTGGCTCTACCAGTGCAGGTCCGCGGTTTGTGAACCAGCATCTGTGCGGCAGCCATCTGGTGGAAGCGCTGTATCTGGTGTGCGGCGAACGCGGCTTCTTTTATACCGATAAAACCCGCGGCATTGTGGAACAGTGCTGCACCAGCATTTGCAGCCTGTATCAGCTGGAAAACTATTGCAACTAA 3’(SEQ ID NO:18)。SEQ ID NO 18的DNA片段是由商业CRO公司化学合成的。一个5’NcoI位点：CCATGG和1个3’Hind III位点：AAGCTT都包含在合成的DNA片段中。将编码SEQ ID NO:14的整个氨基酸序列的DNA片段用NcoI和Hind III限制酶裂解，***到用相同的限制性内切酶裂解的pET 28a表达载体中，形成pET-API表达载体。
将转化的pET-API表达载体转染到BL21(DE3)大肠杆菌中去。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，形成门冬胰岛素原工作种子库(WCB)。
实例2：门冬胰岛素原融合蛋白的表达
将实例1得到的WCB在LB培养基(含1.5％w/v酵母粉和0.5％w/v氯化钠)中37℃下培养8h，得到细胞种子液。种子液被接种到BFM培养基(含0.6％w/v磷酸氢二铵，0.4％w/v氯化铵，1.35％w/w磷酸二氢钾，0.139％w/w 7水硫酸镁，0.28％w/w一水柠檬酸，0.8％w/w葡萄糖，0.3％w/w酵母粉和1％mL/L微量元素溶液)进一步培养8h得到二级种子液，然后按照体积比1：10接种到发酵罐。培养大约16h直到发酵液的OD 600大约为150，然后往发酵罐中加入终浓度为1mM IPTG诱导门冬胰岛素原进行表达。诱导12h，完成发酵过程。通过离心机收集菌体。
将含有门冬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，在15-25℃下溶解10-60min。溶解后的溶液命名为包涵体溶解液。
实例3：门冬胰岛素原的复性
将包涵体溶解液用1μm的PP滤芯进行过滤，将温度控制为20℃，调节pH为11.0，然后进行复性24-48h得到复性的门冬胰岛素原。
实例4：胰蛋白酶酶切转化和纯化
当复性完成后，将复性蛋白溶液pH值调整到9.0。加入终浓度为0.063mg/g蛋白质的胰蛋白酶。在18℃下酶切36h来去除前导肽，以获得B31Arg-门冬胰岛素中间体。这个过程可以由HPLC-RP(C18)进行监控。酶切完成后，溶液中加入终浓度为3mM锌离子，pH值调整为6.0，形成了B31Arg-门冬胰岛素的絮凝沉淀。
B31Arg-门冬胰岛素沉淀用3％异丙醇和50mM醋酸的缓冲液进行溶解，溶解pH值为3.5。溶解的B31Arg-门冬胰岛素作为样品加载到SP层析柱上，用与溶解缓冲液相同的缓冲液平衡。B31Arg-门冬胰岛素可以被30％的异丙醇，1M氯化钠梯度洗脱。通过2次SP层析纯化后，B31Arg-门冬胰岛素的纯度大约是85％。B31Arg-门冬胰岛素洗脱液中加入5mM氯化锌，pH调整到6.5，形成了B31Arg-门冬胰岛素的絮凝沉淀。
进一步的纯化可以用反相制备进行，以除去杂质。最终得到的B31Arg-门冬胰岛素纯度可达98％以上，DesB30-门冬胰岛素含量降至0.1％以下。得到高纯度的B31Arg-门冬胰岛素中间体。
实例5：羧肽酶B酶切转化
将纯化后的B31Arg-门冬胰岛素中间体沉淀用25mM Tris，2mM EDTA进行重悬，调pH为9.0进行溶解。溶解完成后，调节溶液pH为8.5，往溶解液中加入终浓度为0.33mg/g蛋白。在25℃下酶切3h，去除C-肽精氨酸残基得到门冬胰岛素。这个过程可以由HPLC-RP(C18)进行检测。酶切完成后，酶切液的pH值调整为3.8，终止酶切。
实例6：终纯化和冷冻干燥
将转化完成的门冬胰岛素加载到反相制备层析柱上。用0.1M乙酸铵，0.1M乙酸钠，0.1M Tris和pH为7.5的溶液与乙腈按照9:1混合后得到的溶液平衡层析柱。洗脱缓冲液是0.1M乙酸铵，0.1M乙酸钠，0.1M Tris和pH为3.5的溶液与乙腈按照1:9混合后得到的溶液。门冬胰岛素通过线性梯度的洗脱缓冲液洗脱下来。
向反相制备洗脱液中的门冬胰岛素溶液中加入终浓度为5mM的氯化锌，形成沉淀。沉淀用10mM盐酸溶液溶解，控制门冬胰岛素的浓度为8mg/ml。加入终浓度为150mM的NaCl，50mM的柠檬酸三钠，0.5％(v/v)间甲酚和20％(v/v)无水乙醇到门冬胰岛素溶解液溶液中，并将pH值调整到6.3。最后，在溶液中加入终浓度为6mM的乙酸锌。在室温下搅拌约3小时，然后在15℃的温度下放置24小时。结晶完成后通过抽滤来收集晶体。然后用5ml 75％乙醇溶液/(g门冬胰岛素)来清洗晶体，洗涤后收集晶体。然后用10ml乙醇溶液/(g门冬胰岛素)再次清洗晶体，洗涤完成后收集晶体。将晶体转移到真空干燥箱中，在25℃的温度下干燥80h，真空压力不大于-0.08MPa。干燥后得到最终的门冬胰岛素原料药。

Claims (11)

  1. 一种用于制备重组门冬胰岛素及其类似物的新型短门冬胰岛素原序列，其特征在于：包含如公式I所示的氨基酸序列：
    R-R 1-(B 1-B 27)-B 28-B 29-B 30-R 2-(A 1-A 21)
    其中：
    R-R 1是前导肽序列，满足以下条件：
    a.R是超氧化物歧化酶同系物的一部分，它由63个氨基酸组成，包括活性甲硫氨酸；
    2个半胱氨酸残基由丝氨酸取代；
    b.该前导肽不会影响门冬胰岛素原的复性折叠，并且能够被裂解去除；
    c.R 1是精氨酸和赖氨酸中的任意一个；
    R 2是由精氨酸或者赖氨酸中的任意一个构成的C-肽；
    B 1-B 27表示天然人胰岛素B链中B 1-B 27的氨基酸序列；
    A 1-A 21表示天然人胰岛素A链；
    B 28是天冬氨酸，谷氨酸和脯氨酸中的任意一个，优选为天冬氨酸；
    B 29是赖氨酸和脯氨酸中的任意一个，优选为赖氨酸；
    B 30是丙氨酸和苏氨酸中的任意一个，优选为苏氨酸。
  2. 根据权利要求1所述的门冬胰岛素原序列，其特征在于：所述R的序列是SEQ ID NO 1或者SEQ ID NO 2中的一种；
    SEQ ID NO 1序列为：
    MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGP；
    SEQ ID NO 2序列为：
    MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGP。
  3. 根据权利要求1所述的门冬胰岛素原序列，其特征在于：所述R 1和R 2是相同的氨基酸，是精氨酸或赖氨酸中的一种。
  4. 根据权利要求1所述的门冬胰岛素原序列，其特征在于：所述(B 1-B 27)-B 28-B 29-B 30-R 2-(A 1-A 21)是SEQ ID NO 12或者SEQ ID NO 13中的一种；
    SEQ ID NO 12序列为：
    FVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN；
    SEQ ID NO 13序列为：
    FVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQCCTSICSLYQLENYCN。
  5. 根据权利要求1所述的门冬胰岛素原序列，其特征在于：所述门冬胰岛素原序列是SEQ ID NO 14、SEQ ID NO 15、SEQ ID NO 16或SEQ ID NO 17中的一种；
    SEQ ID NO 14序列为：
    MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN；
    SEQ ID NO 15序列为：
    MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPKFVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQCCTSICSLYQLENYCN；
    SEQ ID NO 16序列为：
    MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN；
    SEQ ID NO 17序列为：
    MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPKFVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQCCTSICSLYQLENYCN。
  6. 一种核酸序列，其特征在于：所述核酸序列编码权利要求1所述的门冬胰岛素原序列。
  7. 一种重组表达载体，其特征在于：包含权利要求6所述的核酸序列。
  8. 根据权利要求7所述的重组表达载体，其特征在于：所述的重组表达载体为门冬胰岛素原pET-API。
  9. 一种微生物，其特征在于：包含权利要求7所述的重组表达载体。
  10. 根据权利要求9所述的微生物，其特征在于：所述微生物为包含权利要求8所述的表达载体的大肠杆菌。
  11. 一种门冬胰岛素的制备工艺，其特征在于，包括以下步骤：
    (a)在合适的条件下培养表达具有权利要求1中公式1所述的短门冬胰岛素原序列的大肠杆菌细胞；
    (b)裂解上述培养的大肠杆菌，以提供包含权利要求1所述序列的短门冬胰岛素原的包涵体；
    (c)将包涵体溶解和复性；
    (d)复性完成的门冬胰岛素原用胰蛋白酶酶切转化为门冬胰岛素-R 2中间体粗品；
    (e)用两步离子交换层析和一步制备RP-HPLC层析色谱法对门冬胰岛素-R 2中间体进行纯化，得到高纯度的门冬胰岛素-R 2中间体；
    (f)纯化后的门冬胰岛素-R 2经过羧肽酶B转化，得到成熟的门冬胰岛素；
    (g)羧肽酶B酶切后得到的门冬胰岛素经过制备RP-HPLC反相层析色谱进一步纯化，得到高纯度的门冬胰岛素；
    (h)高纯度的门冬胰岛素经过结晶和干燥形成最终的门冬胰岛素原料药。
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