CN1699412A

CN1699412A - Novel process for genetic engineering preparation of insulin and insulin analogs

Info

Publication number: CN1699412A
Application number: CN 200410018494
Authority: CN
Inventors: 张友尚; 费俭; 丁金国; 崔大敷; 蔡国强; 石嘉豪
Original assignee: SHANGHAI BIO-TAI LIFE SCIENCES RESEARCH Co Ltd
Priority date: 2004-05-20
Filing date: 2004-05-20
Publication date: 2005-11-23
Anticipated expiration: 2024-05-20
Also published as: CN100429226C

Abstract

The invention provides a novel process for genetic engineering preparation of insulin and insulin analogs, which comprises enzyme cutting insulin precursor with parenzyme, then isolating insulin, wherein the insulin precursor comprises the following elements from amino end to carboxyl end, (1) spacing peptide element with formula (Xaa)mZ, (2) insulin B chain element, (3) joint peptide element with formula (Xaa)nZ, and (4) insulin A chain element.

Description

The genetically engineered novel preparation method of Regular Insulin and insulin analog

Technical field

The present invention relates to the physiotechnology field.More specifically, the present invention relates to the new Regular Insulin and the gene engineering preparation method of insulin analog.

Background technology

Regular Insulin is used for the treatment of diabetes from the twenties in 20th century, is still irreplaceable diabetes specifics now.Present global diabetic subject surpasses 1.2 hundred million, and expecting 2025 is 300,000,000, and wherein 10% is type i diabetes.Each type i diabetes patient need use Regular Insulin 1.4-2.1 milligram every day, and the type ii diabetes patient also has sizable demand to Regular Insulin, and developed country will consume 4600 kilograms in Regular Insulin every year.In addition, some new medications can improve the compliance of patient's medication, but bioavailability is low, compares with drug administration by injection, need more Regular Insulin.So in the prevention and control diabetes, the method for researching and developing low-cost mass preparation Regular Insulin is imperative.

The difficult point of preparation Regular Insulin is to form correct paired disulfide linkage at two interchains, the A of Regular Insulin, B chain contain folding full detail, the C-peptide is not to be essential, the connection of C-peptide makes the folding of A, B chain become easy, as intramolecular chaperone, can prevent from the hydrophobic aggregation of Regular Insulin from also to promote to fold as electronegative amino acid.But the isomerization of the Pro that the C-peptide contains makes folding rate slow down.[Chen，L.M.，Yang，X.W.and?Tang，J.G.(2002).Acidic?residues?on?the?N-terminus?of?proinsulinC-Peptide?are?important?for?the?folding?of?insulin?precursor? J?Biochem (Tokyo)131(6)：855-9；Qiao，Z，Feng，Y.(2003)In?vitro?refolding?of?humanproinsulin：kinetic?intermediates，putative?disulfide-forming?pathway，foldinginitiation?site?and?potential?role?of?C-peptide?in?folding?process.J.Biol.Chem.]。Now used clinically insulin human be mainly the intestinal bacteria system and Yeast system expressed.

Escherichia coli expression Regular Insulin is to be cooperated to finish in nineteen eighty-two by Genetech company and Eli Lilly company.Pancreas islet in colibacillary born of the same parents (former) is degraded fast easily, how to make it form inclusion body with fusion rotein now, and also the someone attempts with the intestinal bacteria excreting insulin former.

Have two kinds of common scheme at the expression in escherichia coli pancreas islet: first, difference expressed fusion protein-A chain, fusion rotein-B chain, solubilization of inclusion bodies is after bromize fluoride crack, sulfonated, and Sulfonated A, B chain are folded into Regular Insulin after with ion-exchange or reversed-phase column purifying.This method does not need to use the price comparatively enzyme such as the protaminase [Schmidt of costliness, M., Babu, K.R., Khanna, N., Marten, S.and Rinas, U. (1999) .Temperature-induced production of recombinant human insulin in high-celldensity cultures of recombinant Escherichia coli J Biotechnol68 (1): 71-83.].Second, expressed fusion protein-B chain-C peptide-A chain, be folded into preproinsulin after directly sulfonated behind the solubilization of inclusion bodies, through behind the affinity purification, back formation Regular Insulin and C peptide are handled through trypsinase and protaminase in the alkalescence in the preproinsulin or two basic aminoacidss site.This method can obtain Regular Insulin and C peptide simultaneously.[Jonasson，P.，Nilsson，J.，Samuelsson，E.，Moks，T.，Stahl，S.and?Uhlen，M.(1996).Single-step?trypsin?cleavage?of?a?fusion?protein?to?obtain?human?insulin?and?its?C?peptide Eur?J?Biochem236(2)：656-61；Nilsson，J.，Jonasson，P.，Samuelsson，E.，Stahl，S.and?Uhlen，M.(1996).Integrated?production?of?human?insulin?and?its?C-peptide? J Biotechnol48(3)：241-50]。

Just when Eli Lilly company is devoted to biosynthetic insulin, Novo company produces insulin human [Markussen, J. (1987) .Human insulin by tryptictranspeptidation of porcine insulin and biosynthetic precursors] with the method that enzymatic changes peptide from pork insulin.By 1986, Regular Insulin is secreting, expressing success [Thim, L. in yeast, Hansen, M.T., Norris, K., Hoegh, I., Boel, E., Forstrom, J., Ammerer, G.and Fiil, N.P. (1986) .Secretion and processing of insulinprecursors in yeast Proc Natl Acad Sci U S A83 (18): 6766-70.], make that the pairing of disulfide linkage no longer is the difficult problem of expression of insulin.In this system, proinsulin is secreted in the mode that is similar to aMF.But people also can't use yeast expression system to secrete natural proinsulin [Kjeldsen effectively so far, T. (2000) .Yeast secretory expression of insulin precursors, Appl Microbiol Biotechnol54 (3): 277-86].

Usually used expression strategy is the polypeptide of expressing following structure now: pre-pro-peptide (PreProPeptide)-desB30B chain-miniC peptide-A chain, pre-pro-peptide [Kjeldsen wherein, (the same) 2000], miniC peptide [Kjeldsen, T., Ludvigsen, S., Diers, I., Balschmidt, P., Sorensen, A.R.andKaarsholm, N.C. (2002) .Engineering-enhanced protein secretory expression inyeast with application to insulin J Biol Chem 277 (21): 18245-8] structure all influential to the expression amount of Regular Insulin, shake a bottle expression amount and be generally several mg/litre.In addition, though enzymatic can obtain Regular Insulin after changeing peptide, enzymatic changes the yield that peptide seriously restricts product in industrial production.

In addition, in numerous patent applications such as CN 89108320,89109575.6,90101415.X, 90101564.4,92105143.3,92105888.8,92110456.1,93103997.5,93112839,95115064.2,94193852.2,01110268.3,01140047.1,02136107.X or patent, a large amount of insulin derivates or analogue and corresponding method for making are disclosed.Yet these method for makings mainly are divided into direct chemical synthesis method and method of gene recombination.Chemical synthesis cost height, and method of gene recombination exists shortcomings such as expression amount is low, disulfide linkage mispairing, end product method for making complexity.

Therefore, this area presses for exploitation expression amount height, method for making is easy and Regular Insulin preparation method suitable scale operation.

Summary of the invention

Purpose of the present invention just provides a kind of expression amount height, method for making is easy and Regular Insulin preparation method suitable scale operation.

In a first aspect of the present invention, a kind of insulin precurosor is provided, described precursor contains following element respectively from aminoterminal to carboxyl terminal:

(a) has formula (Xaa) _mSpacer peptide element shown in the Z, in the formula Xaa be in 20 kinds of natural amino acids any, m is the positive integer of 2-10, Z is Arg or Lys;

(b) insulin B chain element;

(c) has formula (Xaa) _nConnection peptides element shown in the Z ', in the formula Xaa be in 20 kinds of natural amino acids any, n is the positive integer of 2-10, Z ' is Arg or Lys;

(d) INSULIN A chain member.

In another preference, described insulin B chain element has following formula:

X ₁?V?N?Q?H?L?C?G?S?H??L?V?E?A?L?Y?L?V?C?G?E?R?X ₂₃?X ₂₄?X ₂₅?X ₂₆?Y ₂₇

Wherein, X ₁For Phe, Ala or there is not X ₂₃, X ₂₄, X ₂₅And X ₂₆Independent separately, be respectively Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Phe, Tyr, Trp, Pro, Met, His, Val or do not exist, and X ₂₃, X ₂₄, X ₂₅And X ₂₆In have one not exist at the most;

Y ₂₇Be Lys, or Arg.

In another preference, X ₂₃X ₂₄X ₂₅X ₂₆Y ₂₇Be GFFYK.

In another preference, the sequence of described spacer peptide element is EAEA (Xaa) _2-4K, in the formula Xaa be in 20 kinds of natural amino acids any, and the sequence of described connection peptides element is (Ala) _2-5Lys.

In another preference, the sequence of spacer peptide element is EAEAYVEFK.

In another preference, also has α-binding factor sequence in the upstream of spacer peptide.

In a second aspect of the present invention, provide a kind of separated DNA, the insulin precurosor that the invention of its code book is above-mentioned.The expression vector that contains described DNA also is provided, and the host cell that contains expression vector or described DNA.Preferably, described host cell is a yeast cell.

In a third aspect of the present invention, a kind of method for preparing Regular Insulin is provided, comprise step:

(a), thereby form Regular Insulin with tryptic digestion insulin precurosor of the present invention;

(b) isolate Regular Insulin.

In another preference, the condition of step (a) is 0-37 ℃, pH4-9, and more preferably the condition of step (a) is 5 ± 4mg/ml insulin precurosor, pH7.5 ± 2, trypsinase: the weight ratio of insulin precurosor is=1: 200 ± 150,30 ± 10 ℃.

In another preference, described insulin precurosor is recombinant expressed, and more preferably, described insulin precurosor is with expressing in the yeast cell (as pichia spp).

Description of drawings

Figure 1A, 1B, 1C have shown that respectively the monomeric insulin precursor (MIP) of three kinds of chemosynthesis (is B ₂₇K-DtrI, B ₁A B ₂₇K-DtrI and (Des B ₁) B ₂₇K-DTrI) gene order and corresponding amino acid sequence figure thereof.

Fig. 2 has shown pPIC9K/MIP plasmid synoptic diagram.

Fig. 3 has shown the optimal ph of shaking table experiment initial optimization fermentation.

Fig. 4 has shown that the cell growth is with the fermentation changing conditions.

Fig. 5 has shown pH8.3 polyacrylamide gel electrophoresis mensuration MIP expression amount, and gum concentration is 15%, Coomassie brilliant blue dyeing.1-5 is respectively from swimming lane: PIP (2 μ g); 24,48,72,84 hours fermented liquids, last sample volume are 20 μ L.

Fig. 6 has shown cationic exchange coloum SP-fast flow purified insulin analogue precursor MIP, initial moving phase is 50mM NaAc-HCl pH4.0 damping fluid, do not adsorb impurity with the initial damping fluid flush away of 5Vc behind the last sample, NaCl concentration increases to 1mol/L from 0 linearity in the moving phase in 15Vc, and 280nm detects.

Fig. 7 has shown SP-fast flow ion exchange column purified insulin analogue B ₂₇K-DTrI, initial moving phase is 50mM NaAc-HCl pH4.0 damping fluid, does not adsorb impurity with the initial damping fluid flush away of 5Vc behind the last sample, and NaCl concentration increases to 1mol/L from 0 linearity in the moving phase in 20Vc, and 280nm detects.

Fig. 8 has shown pH8.3 polyacrylamide gel electrophoresis insulin analog purity, and gum concentration is 15%, Coomassie brilliant blue dyeing.Swimming lane 1-6 is respectively: standard substance (being PIP, Regular Insulin, DOI from top to bottom); The MIP of XAD-7 purifying; The MIP of SP-fast flow ion-exchange column purification; MIP was through TPCK trypsin treatment 30 minutes; MIP was through TPCK trypsin treatment 60 minutes; The B27K-DTrI of SP-fast flow ion-exchange column purification.

Fig. 9 has shown that B27K-DTrI HPLC analyzes, and the C8 post (Beckman, 4.6 * 250mm), Mobile phase B is 70% acetonitrile that contains 0.1%TFA, mobile phase A is the water that contains 0.1%TFA, gradient (30-70%B/10-40min), flow velocity is 1ml/min.X-coordinate is the elution time, and ordinate zou is A280nm.

Figure 10 has shown the electrospray ionization mass spectrum analysis (MATLCQ ESI-MS) of B27K-DTrI, and EFI voltage is 4.25KV, and capillary temperature is 200 ℃.Theoretical molecular is 5508.3, and the actual measurement molecular weight is 5509.0, and error is 0.01%.

Figure 11 shown protein concn to the retention time of chromatography (on) and the influence of peak shape (descending).X-coordinate is a protein concn, and Fs is a symmetrical factor in the ordinate zou, and Kav is a retention factors.

Figure 12 has shown Regular Insulin preparation method's of the present invention synoptic diagram, and wherein arrow is represented restriction enzyme site.

Figure 13 has shown B ₁A B ₂₇K-DTrI and (Des B ₁) B ₂₇The protein concn of K-DtrI is to the influence of the retention time of chromatography, and X-coordinate is a protein concn, and Kay is a retention factors in the ordinate zou.

Figure 14 has shown B ₁A B ₂₇K-DTrI and (Des B ₁) B ₂₇The K-DtrI protein concn to the influence of peak shape, X-coordinate is a protein concn, Fs is a symmetrical factor in the ordinate zou.

Embodiment

The inventor finds at the insulin precurosor shown in the following formula through extensive and deep research:

Spacer peptide element-insulin B chain element-connection peptides element-INSULIN A chain member

Not only can realize high expression level at Yeast system, and help the purifying of purpose product, this precursor also can be through obtaining Regular Insulin once the step enzyme cutting method simultaneously, method for making is very easy, save the enzymatic in the traditional technology and changeed peptide and renaturation step, thereby improved whole productive rate, be particularly suitable for suitability for industrialized production.

As used herein, term " B27K-DTrI " refers to that B chain 27 amino acids are that the B chain carboxylic end of Methionin removes tripeptides Regular Insulin.

As used herein, term " B ₁A B ₂₇K-DtrI " refer to that the 1st on B chain is that the B chain carboxylic end of Methionin removes tripeptides Regular Insulin for Ala and 27 amino acids.

As used herein, term " (Des B ₁) B ₂₇K-DTrI " refer to that B chain the 1st amino acids does not exist and 27 amino acids are that the B chain carboxylic end of Methionin removes tripeptides Regular Insulin.

B27K-DtrI, B ₁A B ₂₇K-DtrI and (Des B ₁) B ₂₇The basic structure of K-DtrI is as follows:

X ₁?V?N?Q?H??L?C?G?S?H??L?V?E?A?L??Y?L?V?C?G??E?R?X ₂₃?X ₂₄?X ₂₅?X ₂₆?Y ₂₇(SEQID?NO：1)

Wherein, X ₁For Phe, Ala or there is not X ₂₃, X ₂₄, X ₂₅And X ₂₆Independent separately, be respectively Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Phe, Tyr, Trp, Pro, Met, His, Val or do not exist, and X ₂₃, X ₂₄, X ₂₅And X ₂₆In have one not exist at the most; Y ₂₇Be Lys, or Arg.Research shows that (can change arbitrarily from the 23rd amino acid) just can have certain activity as long as insulin B chain has preceding 25 amino acid.Therefore, in the present invention, the amino acid of 23-26 position can be arbitrary amino acid, is preferably except Lys any amino acid outside Arg and the Cys, especially L type amino acid.

As used herein, " insulin B chain element " refers to insulin B chain sequence natural or variation.More preferably, described insulin B chain element is that C-terminal amino acid is the insulin B chain or the insulin B chain analogue of basic aminoacids (as Lys or Arg).A kind of preferred example is the element with aminoacid sequence shown in the SEQ ID NO:1.

As used herein, " INSULIN A chain member " refers to INSULIN A chain-ordering natural or variation.A kind of preferred example is the element with the aminoacid sequence shown in the SEQ ID NO:2.

As used herein, " spacer peptide element " refers to be positioned at peptide sequence between homing sequence (as α-binding factor homing sequence) and insulin B chain element, that play interval action.The structure of spacer peptide is (Xaa) _mZ, in the formula Xaa be in 20 kinds of natural amino acids any, m is the positive integer of 2-10, Z is Arg or Lys.Its effect is to provide alkaline Arg or Lys as restriction enzyme site on the one hand; be the expression amount that improves precursor on the other hand; its reason may be to prevent that when expressing the host cell endoenzyme to expressing the enzymolysis of precursor, strengthening the protection to insulin precurosor, thereby improves the precursor quantity of expressing and keeping.Preferred spacer peptide be contain 2 EA at least spacer peptide (as EAEA (Xaa) _2-4K), the spacer peptide of this EA of being rich in is particularly conducive to the secretion of insulin precurosor.A kind of particularly preferred spacer peptide example is EAEAYVEFK (SEQ ID NO:9).[annotate: when with (Xaa) _mWhen being considered as spacer peptide, then the insulin B chain element is that aminoterminal and carboxyl terminal all have insulin B chain or the insulin B chain analogue for basic aminoacids (as Lys or Arg).]

As used herein, " connection peptides element " refers between insulin B chain element and INSULIN A chain member, plays the peptide sequence of ligation.Connection peptides is not particularly limited, and has needed only ligation, and is that Lys or Arg get final product at the amino-acid residue that links to each other with the A chain.One class connection peptides example is (Ala) _2-5Lys, for example Ala-Ala-Lys.

Polypeptide of the present invention can use recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.

Polynucleotide of the present invention can be dna form or rna form.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.

Insulin analog Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

Among the present invention, the insulin analog polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up insulin-containing analogue DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promoter eucaryon promotor comprise CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; Zooblasts such as CHO, COS cell.Particularly preferred host cell is a yeast cell, especially pichia spp.

The expression vector that preferably is used for Yeast system comprises (but being not limited to): pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all can buy) from Invitrogen company.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be handled with the CaCl2 method in exponential growth after date results, and used step is well-known in this area.Another kind method is to use MgCl2.If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

In a preference,, obtain required monomer insulin cutting to handle then through a step enzyme earlier with the monomeric insulin precursor formal representation.To carboxyl terminal, described monomeric insulin precursor contains spacer peptide, insulin B chain, connection peptides and INSULIN A chain respectively from aminoterminal.

Enzyme is cut processing can be with the enzyme of any cutting Lys and/or Arg, for example trypsinase.The enzyme tangent condition can change according to the enzyme of selecting for use.A kind of preferred enzyme tangent condition is: solvent: 0.02-0.05M Tris damping fluid, and pH:7-8, concentration of substrate, about 5mg/ml, the trypsinase consumption is about substrate quality 1/50, and 4-25 ℃, 1-6 hour.Another kind of preferred enzyme tangent condition is 5 ± 4mg/ml insulin precurosor, and pH 7.5 ± 2, trypsinase: the weight ratio of insulin precurosor is=1: 200 ± 150,30 ± 10 ℃, and 1-6 hour.

After the MIP enzyme is cut, separate enzyme with methods such as sieve chromatographies and cut product, can obtain insulin analog of the present invention.

Insulin analog preparation method provided by the invention not only can be used for the preparation of B27K-DTrI, can also be used for the medication preparation of other treatment diabetes insulin class.For example, in CN 89108320,89109575.6,90101415.X, 90101564.4,92105143.3,92105888.8,92110456.1,93103997.5,93112839,95115064.2,94193852.2,01110268.3, numerous patent applications such as 01140047.1 or patent a large amount of insulin derivates or analogue are disclosed.The inventive method has the insulin derivates of insulin activity applicable to these insulin analogs and other.

Major advantage of the present invention is:

(a) insulin precurosor is at the Yeast system high expression level;

(b) insulin precurosor is through obtaining Regular Insulin once the step enzyme cutting method, and method for making is very easy, thereby has improved whole productive rate, is particularly suitable for suitability for industrialized production

(c) need not enzymatic and change peptide or the processing of external renaturation.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The structure of expression vector

The codon usage of pichia spp (P.pastoris) is similar substantially to yeast saccharomyces cerevisiae (S.cerevisiae), but on indivedual amino acid such as Glu, certain difference [Zhao is arranged, X., Huo, K.K.and Li, Y.Y. (2000) .Synonymous codon usage in Pichia pastoris Sheng Wu Gong Cheng XueBao16 (3): 308-11.].According to the codon preference of P.pastoris, chemosynthesis monomer insulin B ₂₇K-DTrI precursor-gene fragment (Figure 1A).

With same procedure chemical synthesis coding B ₁A B ₂₇K-DTrI (Figure 1B) and (DesB ₁) B ₂₇The DNA of K-DTrI (Fig. 1 C).Similar to S.cerevisiae, suitable prepro-leader helps the secreting, expressing of insulin precurosor in P.pastoris, the dna fragmentation of chemosynthesis is cloned into EcoRI, the NotI site of pPIC9K plasmid (Invitrogen company), forms α-Mating factor leader-EAEAYVEFK-MIP and express framework.The spacer peptide (for example EAEAYVEFK, SEQ ID NO:9) that wherein is rich in EA helps the secretion of insulin precurosor, and MIP is B ₂₇K-DTrI precursor, B ₁A B ₂₇K-DtrI precursor or (Des B ₁) B ₂₇K-DTrI precursor, their structure all are insulin B chain 1-26K27AAKA chain 1-21.

It (is pPIC9K/B that the result obtains to contain three kinds of segmental pPIC9K plasmids of MIP respectively ₂₇K-DtrI, pPIC9K/B ₁A B ₂₇K-DtrI or pPIC9K/ (Des B ₁) B ₂₇K-DTrI).For convenience, these three kinds of plasmids are commonly referred to as pPIC9K/MIP (Fig. 2).

Embodiment 2

The screening of plasmid conversion and MIP expression strain

3ug is through the linearizing pPIC9K/B of Bgl II ₂₇K-DtrI plasmid electricity transforms pichia spp GS115 (his4) (available from NRRL, preserving number NRRL Y-15851, US6,730,499), and 0.4cm shock by electricity glass, 2.4KV, and 5.3ms shocks by electricity twice continuously, and transformed yeast cells is coated with the MD plate.Plasmid pPIC9K/B ₂₇After the K-DtrI process Bgl II linearization process, entering the yeast cell rear section can be incorporated in the karyomit(e) by the single-point insertion by recirculation, obtain Mut+ phenotype transformant, a part of in addition linear plasmid is incorporated in the karyomit(e) by replacement, obtain Muts phenotype transformant, in this process, can insert integration by spontaneous formation multiple copied.1500 mono-clonals of picking from the MD plate, 2ml YPD substratum was cultivated 24 hours for 30 ℃, and G418 is 0.5,1,2, the YPD plate of 4mg/ml to containing to get 2 μ L points respectively, and the clone who grows bacterium colony on three blocks of plates enters the phenotypic evaluation experiment.During with methanol yeast expression of insulin precursor, the expression amount of insulin precurosor increases with the increase of the gene copy number that is integrated into yeast chromosomal.Screen the higher transformant of picking gene copy number with G418, the bacterial strain that anti-G418 concentration is 0.5mg/ml and 4mg/ml ferments at 2ml YPM, the expression amount of MIP is respectively 0.5mg/L and 2.5mg/L, and this explanation G418 resistant proof is a kind of method of effectively preliminary screening high expression level bacterial strain really.

From 1500 transformants, select to obtain the clone of 80 anti-G4184mg/ml.The clone of anti-G4184mg/ml is inoculated into MD (1.34%YNB, 4 * 10 simultaneously ^-5The % vitamin H, 2% glucose) plate and MM plate (1.34%YNB, 4 * 10 ^-5The % vitamin H, 0.5% methyl alcohol) on, the clone of no significant difference is the Mut+ phenotype on the speed of growth on the MM plate and MD plate, and poky clone is the Muts phenotype on the MM plate.Contrast experiment by MD plate and MM plate, among the clone of 80 anti-G418 4mg/ml, the Mut+ phenotype accounts for 50%.Because the fermentation period of Mut+ bacterial strain is shorter, select the Mut+ bacterial strain to carry out the small-scale fermentation, choose the higher bacterial strain of secretory volume of MIP.40 Mut+ phenotypic clonings are inoculated into respectively in the 2ml YPD substratum, cultivated 48 hours for 30 ℃, convert the YPM substratum that contains 3% methyl alcohol to, cultivated again 72 hours, use the relative content of measured by radioimmunoassay MIP after supernatant dilutes 400 times with the PBS that contains 0.5%BSA.The expression amount average out to 4mg/L of each bacterial strain, high expression level amount is 4.8mg/L, all can be used for the fermentation of 2L scale.

Embodiment 3

The fermentation of 2L scale

High density fermentation is the effective means that obtains a large amount of heterologous proteins from methanol yeast, and methanol yeast just can be grown in simple minimal medium, and few harmful products such as alcohol and acetate that produce in the fermenting process are so be fit to the large scale and high density fermentation.The growth conditions of fermented liquid pH value pair cell, the stability of secretory protein etc. all have remarkably influenced, with shaking table experiment initial optimization the pH scope of fermentation be 4.5-6.5, optimal ph is 5 (Fig. 3).

(basal salt medium BSM) contains: 50ml glycerine, 26.7mlH every liter of basic minimal medium ₃PO ₄, 0.93g CaSO ₄2H ₂O, 18.2g K ₂SO ₄, 14.9g MgSO ₄7H ₂O) and 4.13g KOH, use NH behind the autoclaving ₄The OH adjust pH.

Trace element solution (PTM1) prescription contains for 1L: 6g CuSO ₄5H ₂O, 0.08g KI, 3.0gMnSO ₄H ₂O, 0.2g (NH ₄) Mo7O ₂₄4H ₂O, 0.02g H ₃BO ₃, 0.5g CaSO ₄2H ₂O, 20gZnSO ₄, 5ml H ₂SO ₄, 65g FeSO ₄7H ₂O, 0.5g CoCl ₂6H ₂O and 0.2g Biotin, filtration sterilization.

To preserve bacterial classification inoculation to 20ml YPG substratum, cultivate 24 hours as first order seed for 30 ℃; The 20ml first order seed is inoculated into 200ml YPG substratum, cultivates OD 10 hours for 30 ℃ _600nmBe about 4, as secondary seed.(fed-batch culture FBC) is divided into three periods: vegetative period to adopt fed batch cultivation; Transitional period; Inductive phase.In 3.7 liters of fermentor tanks, add 8ml PTM1 among the 2L BSM, use NH ₄The OH adjust pH is 5, and temperature is 29 ℃, and keeps 29 ℃ of pH 5.0, temperature during the fermentation, and keeping dissolved oxygen after the inoculation of 200ml secondary seed is 30-35%, begins fermentation.Finish vegetative period after about 20 hours, glycerine exhausts, dissolved oxygen rises suddenly, enter the transitional period (Glycerol fed-batch phase), transitional period is added glycerine with the speed (growth-limiting rate) of limiting growth, feed supplement 50% (W/V) glycerine (containing 0.25%PTM1), and feed supplement speed slowly is raised to 21ml/Lh from 13ml/Lh, each hour measured DO peak shape (spike) and guarantees there is not the glycerine accumulation in the fermented liquid, and dissolved oxygen maintains 30-35%.After 4 hours, change into and mend methyl alcohol (containing 0.2%PTM1), enter inductive phase.Feed supplement speed is 3ml/L.h during beginning, keep dissolved oxygen at 20-30%, feed supplement speed is increased to 12ml/L.h in about 8 hours, and after this dissolved oxygen maintains 20-25%, whole each hour of inductive phase measured the DO peak shape, and the 84 hour cell weight in wet bases of fermenting reach 400mg/ml (Fig. 4).The expression amount average out to 150-250mg/L of insulin precurosor, high expression level amount is 400mg/L.

Embodiment 4

The analysis of expression product and purifying

Get supernatant 20 μ L after fermented liquid is centrifugal, according to Gabriel method [Gabriel O.Analytical disc gelelectrophoresis.Mehtods in Enzymology, 1971,22:656, Jakoby WB.ed, AcademicPress.New York], at the content of pH8.3 polyacrylamide gel electrophoresis analysis MIP.The expression amount of MIP is about 200mg/L, and expression product is two bands (Fig. 5) on the pH8.3 electrophoresis, for spacer peptide in born of the same parents not by due to the complete enzymolysis.Yet this is to not influence of subsequent purification, because unnecessary spacer peptide can be excised by trypsinase in external processing.

Hydrophobic adsorption column XAD-7 (available from Sigma company) has good adsorptive power to MIP, can remove the inorganic salt in the fermented liquid, most polysaccharide and pigment and foreign protein.The XAD-7 post behind 5Vc (promptly 5 times of total column volumes are as follows) methanol wash, 10Vc water balance, the centrifugal cell fermentation liquid upper prop that goes of 10Vc, 10Vc water, 2Vcl 5% ethanol contain 5% acetic acid and wash successively, 60% ethanol contains 5% acetic acid wash-out, 280nm detects.The elutriant rotary evaporation is removed pigment and part polysaccharide with Sephadex G25 after removing ethanol and acetic acid, and moving phase is 1mol/L acetic acid, and 280nm detects.((Amersham Biosciences company) removes impurity such as greasiness class, and (initial moving phase is 50mM NaAc-HCl pH4.0 damping fluid with cationic exchange coloum SP-fast flow to collect protein peak, do not adsorb impurity with the initial damping fluid flush away of 5Vc behind the last sample, NaCl concentration increases to 1mol/L from 0 linearity in the moving phase in 15Vc), 280nm detects, show that the MIP purity of protein that obtains can reach more than 90%, (Fig. 6).

There are 4 basic aminoacids sites in the MIP molecule, because in certain solution environmental, each site molecular conformation difference of living in, trypsinase is different to the kinetics of its effect.In non-optimum condition: 10mg/ml MIP is dissolved in 100mmol/L K ₂HPO ₄-KH ₂PO ₄Damping fluid, pH6.0, TPCK (tosylphenylalanine chloromethyl ketone)-trypsin W): MIP (W)=1: 200, connection peptides Ala-Ala-Lys is observed in 30 ℃ of water-baths ^*, spacer peptide Lys ^*, 27 Lys of B chain ^*All than 22 Arg of B chain ^*Being easier to enzyme cuts.For fear of 22 Arg of chain ^*Place's enzyme is cut and is formed DOI, and excises spacer peptide and connection peptides most effectively, and temperature of reaction, reaction times, pH value, four factors of enzyme-to-substrate ratio have been carried out orthogonal test, and obtain optimal condition and be: 5 ± 4mg/ml MIP is dissolved in 100 ± 50mmol/L K ₂HPO ₄-KH ₂PO ₄Damping fluid, pH7.5 ± 2, TPCK-trypsin W): MIP=1: 200 ± 150,30 ± 10 ℃ were reacted 60 ± 30 minutes, and enzyme is cut productive rate and can be reached more than 95%.

(initial moving phase was 50mM NaAc-HCl pH4.0 damping fluid to mistake SP-fast flow ion exchange column except that foreigh protein removing after endonuclease reaction liquid was transferred the pH4.0 termination reaction with HAc, do not adsorb impurity with the initial damping fluid flush away of 5Vc behind the last sample, NaCl concentration increases to 1mol/L from 0 linearity in the moving phase in 20Vc).280nm detects and shows, obtains the pure product of B27K-DTrI (Fig. 7).

Ultimate yield is 84%.

The evaluation and the activation analysis of embodiment 5 expression products

The B27K-DTrI that purifying obtains is single band (Fig. 8) on the pH8.3 electrophoresis, C8 HPLC analyzes and is unimodal (Fig. 9): C8 reversed-phase column (4.6 * 250mm), mobile phase A is 0.1%TFA, Mobile phase B is 70% acetonitrile that contains 0.1%TFA, gradient (30-70%B/10-40min), flow velocity are 1ml/min.

Through the electrospray ionization mass spectrum determining molecular weight is 5509, and (Figure 10) conforms to theoretical molecular.

Amino acid composition measuring result: representing amino acid with single-letter, is measured value in the bracket; R1 (1.07), K1 (0.9), H2 (2.16), F3 (3.09), Y4 (4.0), L6 (5.6), I2 (1.72), V4 (3.75), A1 (1), G4 (3.7), E and Q7 (7.6), S3 (2.1), T1 (0.82), D and N3 (2.92), C6 (not test).With measure protein concn from external absorbent method: 280nm wavelength, light path 1cm, 1mg/ml Regular Insulin DPI[B28Lys, B29Pro]-the Regular Insulin absorbance value is for being respectively 1.01,0.88,1.01 and 1.07.

The mice convulsion method measures that bioactive method adopts Chinese Pharmacopoeia in the body of B27K-DTrI, and 1985, the method described in 100 pages of the appendix.Method is as follows: male mouse of kunming, body weight are 27-30g, divide four groups at random by body weight, and 24 every group, fasting is 2 hours before the experiment.It is 5 physiological saline solution that sample transfers to the pH value with hydrochloric acid, 1OD280nm=1mg/L.Sample estimates are tired and are 80% of standard substance, and high dosage is 0.09U/ml, and low dosage is 0.045U/ml, and solvent is that hydrochloric acid adjusting pH is 2.5 physiological saline.25 ℃, every mouse subcutaneous injection 0.3ml in 15 minutes places 37 ℃ to observe 90 minutes, and is convulsions, dead, it is lain on the back can stand up the person voluntarily in back 3 seconds and all think positive reaction.Data are calculated by the qualitative response assay method.The result shows, B ₂₇In the body of K-DtrI biological activity be Regular Insulin 80% (the mice convulsion method records B ₂₇The activity of K-DtrI is 21U/mg, and insulin standard product 27U/mg).

The monomer property testing of B27K-DTrI, in neutral solution, form the mixture of monomer, disome, hexasomic after the polymerization of higher concentration Regular Insulin, use the gathering character of FPLC gel filteration determining dezincify Regular Insulin, Superdex 75 (HR 10/30) post, moving phase be PBS (phosphate-buffered saline, pH7.4), flow velocity is 0.4ml/min, applied sample amount is 0.1ml, room temperature, and 280nm detects.Rising along with the dezincify insulin concentration, its retention time on Superdex 75 (HR 10/30) post shortens, asymmetry of peak strengthens, we describe this variation with Kav and Fs respectively, all once describe mixture with symmetrical factor Fs, describing the molecular-weight average of mixture with partition ratio Kav.Fs=W0.05h/2A, wherein W0.05h is the peak width at 0.05 peak height place, A is the preceding peak width at half height at 0.05 peak height place; Kav=(Vr-Vo)/(Vc-Vo), wherein Vr is an efflux volume, and Vo is a void volume, and Vc is total column volume.Under identical condition, compared the situation (Figure 11 A and B) that Kav, the Fs of dezincify Regular Insulin, DPI, [B28Lys, B29Pro]-Regular Insulin and B27K-DTrI change with protein concentration.DPI, [B28Lys, B29Pro]-Regular Insulin and B27K-DtrI are monomer insulins known in the art, and their monomer character has tangible difference, and wherein DPI is the strongest, and B27K-DTrI slightly is better than [B28Lys, B29Pro]-Regular Insulin.

Same technological line also can be used for expressing other other yeast expression system or secretory coli expression system of insulin analog of preparation and expresses B27K-DTrI.This technological line is compared with the preparation method of disclosed DTI in the Chinese patent application 98110912.8, not only can avoid the little peptide GFFY of chemosynthesis (But) Obut, enzymatic changes peptide, loaded down with trivial details technologies such as HPLC separation, and can further significantly improve the expression amount of precursor, thereby the raising productive rate is more suitable in industrial production.Enzyme cutting process synoptic diagram of the present invention is seen Figure 12.

Embodiment 6

B ₁A B ₂₇K-DtrI and (Des B ₁) B ₂₇The preparation of K-DtrI

The method identical with embodiment 2-4 prepares B ₁A B ₂₇K-DtrI and (Des B ₁) B ₂₇K-DtrI, difference only are the pPIC9K/B with preparation among the embodiment 1 ₁A B ₂₇K-DtrI or pPIC9K/ (Des B ₁) B ₂₇K-DtrI replaces pPIC9K/B ₂₇K-DtrI.The result has obtained B equally ₁A B ₂₇K-DtrI and (Des B ₁) B ₂₇K-DtrI.

B ₁A B ₂₇K-DTrI and (Des B ₁) B ₂₇Biological activity is 80% (the mice convulsion method records two kinds of monomeric activity and is 22U/mg, and insulin standard product 27U/mg) of Regular Insulin in the body of K-DtrI.

Simultaneously, under above-mentioned identical condition, dezincify Regular Insulin, B have been compared ₁A B ₂₇K-DTrI and (DesB ₁) B ₂₇The situation that the Kav of K-DTrI, Fs change with protein concentration.Result such as Figure 13 and shown in Figure 14 show B ₁A B ₂₇K-DTrI and (Des B ₁) B ₂₇K-DTrI has significant monomer character.

In addition, immunogenicity determining also shows B ₁A B ₂₇K-DTrI and (Des B ₁) B ₂₇The immunogenicity of K-DTrI has obvious decline.

Embodiment 7

The spacer peptide effect relatively

B with the identical no spacer peptide of method preparation of embodiment 2-4 ₂₇The encoding sequence of K-DtrI precursor and spacer peptide are the B of YVEFK ₂₇K-DtrI precursor precursor encoding sequence changes pichia spp then equally over to, and the expression of insulin precursor is used the tryptic digestion precursor again, and separation and purification obtains B then ₂₇K-DtrI.Three kinds of B ₂₇The situation of K-DtrI precursor relatively sees the following form.

	Spacer peptide	The secreting, expressing amount	The separation and purification effect
	Spacer peptide	The secreting, expressing amount	The separation and purification effect	Comparative Examples	Do not have	Generally	The impurity that has the excision of N end parts, extremely difficult the removal, purification effect is poor
The present invention	?YVEFK	Higher	Impurity is easily removed, and purification effect is good	Comparative Examples	Do not have	Generally
	?YVEFK	Higher	Impurity is easily removed, and purification effect is good	?EAEAYVEFK	Higher	Impurity is easily removed, and purification effect is good

The above results shows, introduce the secretion that spacer peptide not only helps insulin precurosor, and because the existence of spacer peptide, the N end that can prevent the insulin B chain element is by part excision (as excising 2 or more a plurality of amino acid), thereby avoided forming and required product characteristics impurity very approaching, that be difficult to remove, so the separation and purification effect of enzyme after cutting is splendid.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉go up the safe life science of marine life company

＜120〉the genetically engineered novel preparation method of Regular Insulin and insulin analog

<130>043293

<160>9

<170>PatentIn?version?3.1

<210>1

<211>27

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(23)..(26)

<223>Xaa＝Gly，Ala，Asp，Glu，Asn，Gln，Ser，Thr，Leu，Ile，Lys，Arg，P

He, Tyr, Trp, Pro, Cys, Met, His, or Val

<220>

<221>MISC_FEATURE

<222>(1)..(1)

＜223〉Xaa=Phe, Ala or nothing

<220>

<221>MISC_FEATURE

<222>(27)..(27)

＜223〉Xaa=Lys or Arg

<220>

<221>MISC_FEATURE

<222>(1)..(27)

＜223〉insulin B chain

<400>1

Xaa?Val?Asn?Gln?His?Leu?Cys?Gly?Ser?His?Leu?Val?Glu?Ala?Leu?Tyr

1???????????????5???????????????????10??????????????????15

Leu?Val?Cys?Gly?Glu?Arg?Xaa?Xaa?Xaa?Xaa?Xaa

20??????????????????25

<210>2

<211>21

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(21)

＜223〉INSULIN A chain

<400>2

Gly?Ile?Val?Glu?Gln?Cys?Cys?Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu

1???????????????5???????????????????10??????????????????15

Glu?Asn?Tyr?Cys?Asn

20

<210>3

<211>173

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(173)

＜223〉monomer insulin B27K-DTrI precursor encoding sequence

<400>3

gaattcaagt?tcgtcaacca?acacttgtgt?ggttcccact?tggtcgaggc?tttgtacttg?????60

gtctgtggtg?aaagaggttt?cttctacaag?gctgctaagg?gtatcgtcga?acaatgttgt?????120

acctccatct?gctccttgta?ccaattggag?aactactgta?actaggcggc?cgc????????????173

<210>4

<211>51

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(51)

＜223〉monomer insulin B27K-DTrI precursor

<400>4

Phe?Val?Asn?Gln?His?Leu?Cys?Gly?Ser?His?Leu?Ala?Glu?Val?Leu?Tyr

1???????????????5???????????????????10??????????????????15

Leu?Val?Cys?Gly?Glu?Arg?Gly?Phe?Phe?Tyr?Lys?Ala?Ala?Lys?Gly?Ile

20??????????????????25??????????????????30

Val?Glu?Gln?Cys?Cys?Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu?Glu?Asn

35??????????????????40??????????????????45

Tyr?Cys?Asn

50

<210>5

<211>173

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(173)

＜223〉monomer insulin B1A B27K-DTrI precursor encoding sequence

<400>5

gaattcaagg?ctgtcaacca?acacttgtgt?ggttcccact?tggtcgaggc?tttgtacttg?????60

gtctgtggtg?aaagaggttt?cttctacaag?gctgctaagg?gtatcgtcga?acaatgttgt?????120

acctccatct?gctccttgta?ccaattggag?aactactgta?actaggcggc?cgc????????????173

<210>6

<211>51

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(51)

＜223〉monomer insulin B1A B27K-DTrI precursor

<400>6

Ala?Val?Asn?Gln?His?Leu?Cys?Gly?Ser?His?Leu?Ala?Glu?Val?Leu?Tyr

1???????????????5???????????????????10??????????????????15

Leu?Val?Cys?Gly?Glu?Arg?Gly?Phe?Phe?Tyr?Lys?Ala?Ala?Lys?Gly?Ile

20??????????????????25??????????????????30

Val?Glu?Gln?Cys?Cys?Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu?Glu?Asn

35??????????????????40??????????????????45

Tyr?Cys?Asn

50

<210>7

<211>170

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(170)

＜223〉monomer insulin (Des B1) B27K-DTrI precursor encoding sequence

<400>7

gaattcaagg?tcaaccaaca?cttgtgtggt?tcccacttgg?tcgaggcttt?gtacttggtc??????60

tgtggtgaaa?gaggtttctt?ctacaaggct?gctaagggta?tcgtcgaaca?atgttgtacc?????120

tccatctgct?ccttgtacca?attggagaac?tactgtaact?aggcggccgc????????????????170

<210>8

<211>50

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(50)

＜223〉monomer insulin (Des B1) B27K-DTrI precursor

<400>8

Val?Asn?Gln?His?Leu?Cys?Gly?Ser?His?Leu?Ala?Glu?Val?Leu?Tyr?Leu

1???????????????5???????????????????10??????????????????15

Val?Cys?Gly?Glu?Arg?Gly?Phe?Phe?Tyr?Lys?Ala?Ala?Lys?Gly?Ile?Val

20??????????????????25??????????????????30

Glu?Gln?Cys?Cys?Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu?Glu?Asn?Tyr

35??????????????????40??????????????????45

Cys?Asn

50

<210>9

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(9)

＜223〉spacer peptide

<400>9

Glu?Ala?Glu?Ala?Tyr?Val?Glu?Phe?Lys

1???????????????5

Claims

1. an insulin precurosor is characterized in that, described precursor contains following element respectively from aminoterminal to carboxyl terminal:

(b) insulin B chain element;

(d) INSULIN A chain member.

2. precursor according to claim 1 is characterized in that described insulin B chain element has following formula:

X ₁VNQHLCGSH?LVEAL?YLVCG?ERX ₂₃X ₂₄X ₂₅X ₂₆Y ₂₇

Y ₂₇Be Lys, or Arg.

3. precursor as claimed in claim 2 is characterized in that X ₂₃X ₂₄X ₂₅X ₂₆Y ₂₇Be GFFYK.

4. precursor according to claim 1 is characterized in that the sequence of described spacer peptide element is EAEA (Xaa) _2-4K, in the formula Xaa be in 20 kinds of natural amino acids any, and the sequence of described connection peptides element is (Ala) _2-5Lys.

5. precursor as claimed in claim 1 is characterized in that, the sequence of spacer peptide element is EAEAYVEFK, and also has α-binding factor sequence in the upstream of spacer peptide.

6. a separated DNA is characterized in that, the described insulin precurosor of its coding claim 1.

7. an expression vector is characterized in that, it contains the described DNA of claim 6.

8. a host cell is characterized in that, it contains the described expression vector of claim 7 or contains the described DNA of claim 6.

9. a method for preparing Regular Insulin is characterized in that, comprises step:

(a), thereby form Regular Insulin with the described insulin precurosor of tryptic digestion claim 1;

(b) isolate Regular Insulin.

10. method as claimed in claim 9 is characterized in that, the condition of step (a) is 5 ± 4mg/ml insulin precurosor, pH7.5 ± 2, and trypsinase: the weight ratio of insulin precurosor is=1: 200 ± 150,30 ± 10 ℃.