CN102558356A

CN102558356A - Human proinsulin fusion protein and preparation method of human insulin

Info

Publication number: CN102558356A
Application number: CN2010106219476A
Authority: CN
Inventors: 傅海龙; 李忠秀; 孙祥明; 包骏
Original assignee: Shanghai Kexin Biotech Co Ltd
Current assignee: Shanghai Kexin Biotech Co Ltd
Priority date: 2010-12-31
Filing date: 2010-12-31
Publication date: 2012-07-11

Abstract

The invention belongs to the technical field of the gene engineering, and particularly relates to a human proinsulin fusion protein and a preparation method of human insulin. The human proinsulin fusion protein comprises X-B-C-A four peptide chains from an N terminal to a C terminal, wherein the X peptide chain is deleted or a guide peptide, the B-C-A peptide chains have amino acid sequences shown by SEQ ID NO.3 or 90% homologous with amino acid sequences shown by SEQ ID NO.3 and mature body of the human proinsulin fusion protein comprises mutant with hypoglycemic activity, and is characterized in that: the Lys 64 residues on the C terminal of the C peptide are substituted by non basic amino acids. In the human proinsulin fusion protein, Arg-A0 insulin by-products formation is avoided, wherein the C peptide can promote the proinsulin renaturation. The preparation method has the following advantages: relatively less required steps, relatively less by-products and relatively high yield of the insulin.

Description

A kind of proinsulin human's fusion rotein and insulin human's preparation method

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of proinsulin human's fusion rotein and insulin human's preparation method.

Background technology

Regular Insulin is a kind of nonglycosylated heterologous polypeptide dimer that is connected by disulfide linkage; Form by 51 amino acid altogether; Be respectively 21 amino acid whose A peptides and 30 amino acid whose B peptides; Two pairs of disulfide linkage by between A7-B7 and the A20-B19 connect, and also have the intrachain disulfide bond of an A6-A11 in addition at the A peptide.(Fig. 1)

Regular Insulin is synthetic with insulinogenic form by pancreatic beta cell, and the C of B peptide end is connected to a strand through an other peptide chain C peptide and A peptide N in the proinsulin.The C peptide contains 35 amino acid altogether, and its N end is Lys-Arg (KR) for Arg-Arg (RR) C end.Sophisticated Regular Insulin is that the enzymatic lysis by β emiocytosis forms, have three kinds of enzymes to participate in this process: PC1/3 and distinguish cracking B-C junction and C-A junction with PC2, with the C peptide at first cracking remove; The unnecessary Arg of the B peptide C end that the then responsible cracking of carboxypeptidase H produces when the PC1/3 cracking is to form sophisticated Regular Insulin.

Treatment is divided into following several kinds with the working method of Regular Insulin: 1, extract from animal pancreas, like pork insulin, zoogenous Regular Insulin also occupies very big share on the Chinese Medicine market at present, is the listed kind of national essential drugs catalogue; 2, chemosynthesis; 3, semi-synthetic: as to be about to pork insulin and to be converted into Asn, form the insulin human through the chemical action Ala that its A peptide is terminal; 4, recombinant protein fermentation expression.

The insulin human is first kind of recombinant protein medicine that FDA ratified, in recent years, recombinant expressed preparation insulin human by successful Application in various host, comprise bacterium, yeast, zooblast even plant.But its ultimate principle is nothing more than following three kinds:

1, double-stranded method:

The scientists in the city that the recombinant human insulin is hoped by the U.S. is the earliest come company in 1978 successfully preparations and authorized gift as first gene recombinant protein medicine by FDA in nineteen eighty-two.Initial method be with A peptide and B peptide gene respectively with the sweet enzyme of the beta galactose (gene splicing of β-gal); Fusion gene transforms different intestinal bacteria and independently ferments; The inclusion body that two expressed fusion roteins form in cell is folding to obtain sophisticated insulin human (Chance et al. through A, the B two peptide vitro recombination of renaturation, the cracking of cyaniding bromine, generation; Diabetes Care, 1981,4).Yet; The sweet enzyme molecular weight of beta galactose is big (～1000 amino acid); Cause the insulin peptide ratio in the tunning on the low side, the sweet enzyme of later stage beta galactose is replaced by one the about 190 amino acid whose polypeptide (Trp-LE) of tryptophan operon, thereby makes the mature insulin productive rate greatly promote.

In the actually operating, owing to need twice different fermenting process, and A, B two peptides after Trp-LE-Met-A fusogenic peptide and the cracking of Trp-LE-Met-B fusogenic peptide cyaniding bromine need carry out sulfonation protection, have obviously improved production cost.Therefore, present this method has not been used further to produce.

2, proinsulin method (inclusion body in the born of the same parents):

A kind of in addition method be with proinsulin as an expression of polypeptides, this method has been eliminated fermenting twice necessary in the double-stranded method.Its basic implementation method is carried out the fusion rotein fermentation expression for a guiding peptide (like Trp-LE-Met) is merged at proinsulin N end.(Kroeff et al.; J.Chromatogr.1989; 46) behind the solubilization of inclusion bodies that fermentation produces; At first by cyaniding bromine excision guiding peptide, the renaturation under sulfonation is protected of proinsulin afterwards is folding, and correct folding product can use trypsinase and protaminase cracking and process multistep chromatography to obtain the Regular Insulin end product.(Kemmler?et?al.，J.Biol.Chem.，1971，246)

Trp-LE-Met is as a fusion tag, and its molecular size (～190 amino acid) is compared still bigger, final Regular Insulin amino acid accounting less than 20% of leavened prod with proinsulin (86 amino acid).Meanwhile, use the processing of the toxic substance that the mode of cyaniding bromine produces and the control of cyaniding bromine more increased cost to aforesaid method.Therefore, shorter and guiding peptide that have the proteolytic enzyme recognition site is proposed in succession.Met-Tyr such as USP 5126249; 5378613 Met-Arg and Chen et al.; (Appl.Biochem.Biotechnol.1999; 55) Met-Lys and the USP 5358857 described, Korean Patent (Registration#1002029580000), Tikhonov et al. (Prot.Exp.Purif.; Many novel guiding peptides 2002,26) etc. have been described.The proinsulin expression vector that contains these guiding peptides has the high expression level amount more, but in the late enzyme cracking is handled, can produce a large amount of by products, makes the purifying work in downstream become difficult.Chinese patent 98813941.3 has been described with human growth hormone as the guiding peptide; And the expression vector that links to each other with proinsulin and form via Arg or Lys, but its Regular Insulin amino acid no accounting is not high, and enzymatic lysis efficient is lower; Renaturation process need consume a large amount of ureas, and production cost increases.

3, proinsulin method (secreting solvable):

A common ground of double-stranded method and inclusion body proinsulin method is that the fusion rotein tunning exists with the inclusion body form.Inclusion body is a kind of improper folding aggregate, and its generation is too high can not the folding timely of expression speed that comparatively fast causes foreign protein owing to intestinal bacteria rrna translation speed.Inclusion body is generally soluble, needs to carry out renaturation work after the redissolution of use high density denaturing agent, and purifying process is complicated.

For Regular Insulin, because it has 3 pairs of disulfide linkage, in the Escherichia coli cell of relative reducing environment, can't form the disulfide linkage of correct pairing, more aggravated the formation of inclusion body.Therefore, Regular Insulin generally all carries out to be secreted into the cell pericentral siphon at colibacillary solubility expression.Because the independent proinsulin of expressing is extremely lacked (Chan et al., PNAS, 1981,78) in the transformation period of cell pericentral siphon, therefore general soluble-expression all is to carry out with the fusion rotein form simultaneously.(Malik?et?al.，Prot.Exp.Puri.，2007，55；Winteret?al.，J.Biotech.，2000，84；Kang?and?Yoon，J.Biotech，1994，36)。Yet the colibacillus periplasm space is less, and the inclusion body of expressing in expressed fusion amount of insulin that goes out and the born of the same parents differs greatly, and has limited its application aborning.Then be to be host cell (Thim et al., PNAS, 1986,83.) in the actual production, correct renaturation proinsulin is secreted in the substratum with cereuisiae fermentum (Saccharomyces cereviseae).The secretion gained contains the proinsulin of disulfide linkage of correct pairing through after changeing peptide and trypsinase cracking, behind chromatography, obtains end product.Though but yeast expression system downstream post-treatment is simpler than bacterial expression system, its shortcoming is that growth is slow, and the production cycle is longer, and output is lower, cost is high.

Summary of the invention

Problems such as Arg-A0 Regular Insulin by product that exists in the technical problem prior art to be solved by this invention and renaturation.

For this reason; The invention discloses a kind of proinsulin human's fusion rotein; Hold to C end from N and to form by four sections peptide chains of X-B-C-A; Wherein said X peptide chain is not for having or the guiding peptide, said B-C-A peptide chain for its aminoacid sequence shown in SEQ ID NO.3 or for have the two mutants of hypoglycemic activity at least with aminoacid sequence 90% homology shown in the SEQ ID NO.3 and its ripe body, it is characterized in that the Lys64 residue of said C peptide C end is replaced by non-basic aminoacids.Said non-basic aminoacids is Ser, Gly or Val.

In an embodiment preferred of this proinsulin human's fusion rotein, the aminoacid sequence of said C peptide is shown in SEQ ID NO.4.

In another embodiment preferred of this proinsulin human's fusion rotein, the aminoacid sequence of said C peptide is shown in SEQ ID NO.5.

In the another embodiment preferred of this proinsulin human's fusion rotein, the aminoacid sequence of said C peptide is shown in SEQ ID NO.6.

In a preferred embodiment again of this proinsulin human's fusion rotein, said guiding peptide holds the C end to comprise from N: the first peptide section follows 6-10 Histidine closely by methionine(Met); The second peptide section is that 1-3 glycocoll-Serine repeats to follow closely Methionin or l-arginine.

In again again in the preferred embodiment of this proinsulin human's fusion rotein, the said first peptide section aminoacid sequence is shown in SEQ ID NO.1.

In in addition again in the preferred embodiment of this proinsulin human's fusion rotein, the said second peptide section aminoacid sequence is shown in SEQ ID NO.2.

In a most preferred embodiment again of this proinsulin human's fusion rotein, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

In a most preferred embodiment again of this proinsulin human's fusion rotein, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

In a highly preferred embodiment again of this proinsulin human's fusion rotein, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

On the other hand, the invention also discloses a kind of nucleotide sequence of the above-mentioned proinsulin human's fusion rotein of encoding.

In one of this nucleotide sequence most preferred embodiment, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

In another most preferred embodiment of this nucleotide sequence, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

In another highly preferred embodiment of this nucleotide sequence, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

On the other hand, the invention discloses a kind of recombinant vectors that contains the above-mentioned proinsulin human's fusion rotein nucleotide sequence of encoding.

In a most preferred embodiment of this recombinant vectors, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

In another most preferred embodiment of this recombinant vectors, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

In another highly preferred embodiment of this recombinant vectors, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

In another highly preferred embodiment of this recombinant vectors, said recombinant vectors is the pCRT7NT plasmid.

On the other hand, the invention also discloses a kind of transformant that contains the recombinant vectors of the above-mentioned proinsulin human's fusion rotein nucleotide sequence of encoding.

In a most preferred embodiment of this transformant, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

In another most preferred embodiment of this transformant, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

In the another most preferred embodiment of this transformant, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

In a highly preferred embodiment again of this transformant, the aminoacid sequence of said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

On the other hand, the invention discloses a kind of insulin human's preparation method, comprise the following steps:

A) make up the recombinant vectors that contains the above-mentioned proinsulin human's fusion rotein nucleotide sequence of encoding;

B) obtain transformant;

C) fermentation;

D) results contain the tunning part of proinsulin human's fusion rotein;

E) renaturation;

F) purifying protein;

G) trypsinase cracking: through the 5KD ultrafiltration, pH10-11 keeps purifying protein concentration at 8-12mg/ml, adding trypsinase at 1: 1000,37 ℃ cracking 3-4 hour, add phosphoric acid to the pH-3.5 termination reaction, remove trypsinase;

H) protaminase cracking: through the 5KD ultrafiltration, pH8.0 adds 1: 1000 protaminase cracking 2 hours, adds phosphoric acid to pH 3.5 termination reactions, removes protaminase;

I) albumen recrystallization promptly obtains the insulin human.

In some embodiment of above-mentioned preparation method, among the said step e renaturation be sex change proinsulin human's fusion rotein with contain mercaptan, oxidized form mercaptan dimer contacts with denaturant solution.

Said proinsulin human's fusion rotein concentration is at 0.3-2mg/ml; Said denaturing agent is selected from urea or/and Guanidinium hydrochloride wherein is preferably urea, and urea concentration is 0.5-1.5M.

The pH of renaturation is 9-11 among the said step e, and wherein preferred pH value scope is 10.5-11.0.Mercaptan and the dimeric ratio of oxidized form mercaptan are 1: 1-1: 10, mercaptan be chosen as halfcystine, oxidized form mercaptan dimer is a Gelucystine, wherein the ratio of halfcystine and Gelucystine is 1: 5-1: 7.

Proinsulin human's fusion rotein according to the invention has been avoided Arg-A0 Regular Insulin production of by-products, and wherein the C peptide can promote insulinogenic renaturation.The required step of said method is less, and it is less to produce by product, obtains the Regular Insulin of high yield.

Description of drawings

Fig. 1, insulin human's structure that the disulfide linkage pairing is correct.

Fig. 2, His-K64S proinsulin expression vector (V3phi) signal collection of illustrative plates.

Fig. 3, mutant and wild-type rely auxilliary proinsulin expression pattern: swimming lane 1 is a protein molecular weight standard, and swimming lane 2 is not for inducing contrast, and swimming lane 3 is the K64S mutant; Swimming lane 4 is the K64G mutant; Swimming lane 5 is the K64V mutant, and swimming lane 6 is a wild-type, and swimming lane 7 is the proinsulin renaturation product.

The HPLC that enzyme is cut product after Fig. 4, the His-K64S proinsulin renaturation analyzes, peak 6.7,8.2, and 9.3,9.8 minutes are corresponding is each peak of trypsinase single endonuclease digestion product, the peak corresponded to Regular Insulin behind the purifying in 10.0 minutes.Use moving phase to be A phase 0.1%TFA, 10% acetonitrile; B phase 0.1%TFA, 100% acetonitrile.10 minutes gradient time.

Fig. 5, bioactivity research, curve are shown injecting normal saline (■), the new Insulin lispro of section (●) and gift respectively and come excellent happy (▲) back rabbit blood glucose concentration changes with time of secreting.

Embodiment

Expression of Fusion Protein

At this paper, the disclosed fusion rotein of the present invention can obtain in several ways, perhaps in proper host cell, expresses such as chemosynthesis.The gene of fusion rotein disclosed by the invention of encoding can be cloned in the carrier that is fit to different cells, including, but not limited to pUC, pBlueScript, pBR322, pMD etc. through different methods.Gene clone can realize through using conventional cloning process; Be connected in the carrier with identical restriction enzyme site such as utilizing restriction enzyme to shear; Perhaps be inserted in the linearized vector, and use the T-A cloning process to be inserted in the T carrier through the PCR product.

Said fusion rotein of the present invention is proinsulin human and two mutants thereof; Its functional activity fragment or its analogue; The proteinic carrier that homologue and coding with high homology comprises the aminoacid sequence that SEQ ID NO.3 describes is dna vector (plasmid or virus) for example.Functional activity fragment or analogue can form through one or more amino-acid residue that adds, inserts, modifies, replaces or lack in the above listed aminoacid sequence.

Term " analogue " also comprises precursor and other functional equivalent or the stand-in of chimeric protein, fusion rotein, antiidiotypic antibody, above-claimed cpd.Also comprise the conjugated protein active compound body of Simulation with I L-1Ra.

Term " two mutants " is meant aminoacid sequence such as the said proinsulin human's of SEQ ID NO.3 two mutants.Than natural proinsulin human, this two mutants is compared with their wild-types, has the active and/or altered stereospecificity of enhanced.The aminoacid sequence two mutants of native protein can be through introducing suitable Nucleotide variation or preparing through external synthetic required polypeptide in Nucleotide of the present invention.These two mutants comprise, for example lack, insert or replace the residue in this aminoacid sequence.Can through disappearance, insert and the combination of replacement to obtain final construct, final protein product is provided.

The nucleic acid of the novel C peptide of code book Invention Announce can be generated by rite-directed mutagenesis; The method of rite-directed mutagenesis has multiple; Comprise mega primer method, inverse PCR method etc. can be bought corresponding reagent box (like Stratagene Quik Change) or used high-fidelity DNA polymerase to carry out by commerce.

Term " ripe body " be meant proinsulin human and two mutants thereof through enzyme cut or modification or conformational change after the albumen of biologically active, the present invention refers to insulin human and two mutants thereof especially.

Proteic percent homology is analyzed (GCG program) by GAP (Needleman and Wunsh, 1970) and is confirmed, parameter gap creation penalty=5 wherein, gap extension penalty=0.3.When the sequence length of being analyzed was at least 15 amino acid, GAP just analyzed and tests in 15 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 50 amino acid, GAP just analyzed and tests in 50 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 100 amino acid, GAP just analyzed and tests in 100 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 250 amino acid, GAP just analyzed and tests in 250 the amino acid whose zones that are at least of two sequences of participating in test.Even more preferably, when the sequence length of being analyzed was at least 500 amino acid, GAP just analyzed and tests in 500 the amino acid whose zones that are at least of two sequences of participating in test.

The aspect that the present invention relates to also comprises proinsulin human's analogue; Carrying out different modifications between their synthesis phases or after synthetic; For example, through biotinylation, benzylization, glycosylation, acetylize, phosphorylation, by known protection/blocking groups derivatization, proteoclastic cutting action, be connected to antibody molecule or other cell ligand is first-class.These modifications can be used for increasing insulinogenic stability of the inventor and/or biological activity.

The present invention includes coding proinsulin human's of the present invention DNA and carrier, the transformant that contains these DNA.

Among the present invention, the term of use " transformant " (transformant) promptly has the host cell of allogeneic dna sequence DNA molecule.

The present invention also comprises through synthetic and produces proteic method according to the invention with recombinant technology.Can separate and purifying polynucleotide (DNA or RNA), carrier, transformant and organism through methods known in the art.

Being used for carrier of the present invention can be like phage, plasmid, clay, minichromosome, virus or retroviral vector.The carrier that can be used for cloning and/or express polynucleotide of the present invention is to duplicate and/or to express the carrier that duplicates and/or express polynucleotide in the host cell of polynucleotide at need.Generally speaking; Polynucleotide and/or carrier can be used for any eucaryon or prokaryotic cell prokaryocyte, comprise mammalian cell (like people (like HeLa), monkey (like Cos), rabbit (like the rabbit reticulocyte), rat, hamster (like CHO, NSO and baby hamster kidney cell) or mouse cell (like the L cell)), vegetable cell, yeast cell, insect cell or bacterial cell (like intestinal bacteria).Relevantly be applicable to that the example of the suitable carrier of broad variety host cell can be referring to for example F.Ausubel et al., Current Protocols in Molecular Biology.Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al. (1989).Can use the host cell that contains these polynucleotide to come great expression to can be used for the for example protein of medicine, diagnostic reagent, vaccine and therapeutical agent.

Having developed several different methods is used for via the complementary sticky end polynucleotide being operated with carrier and links to each other.For example, can add complementary with the aggressiveness sequence fragment by the DNA section in desire is inserted carrier DNA.Then through the hydrogen bond connection carrier between the complementary homopolymeric tail and DNA section to form recombinant DNA molecules.The insertion of the recombinant DNA of required expression can be identified through some ordinary methods: 1) identify that through PCR size that recombinant bacterial strain is inserted into the zone inserts segmental empty carrier respective regions size relatively with not having; 2) through the big or small variation of carrier; 3) insert segmental expression.

The synthetic linker that contains one or more restriction site provides the method for another kind of connection DNA section and carrier.Handle the DNA section that produces through the endonuclease restrictive diges-tion with phage T4DNA polysaccharase or e. coli dna polymerase I; Described two kinds of polysaccharases with its 3 '; It is terminal that 5 '-exonucleolytic activity is removed outstanding γ-strand, and mend flat 3 '-recessed end with its polymerization activity.Therefore, these active associatings have produced flush end DNA section.The enzyme that connects at ability catalysis flush end dna molecular then is as under the existence of phage T4DNA ligase enzyme being incubated the linkers of flush end section with molar excess.Therefore, reaction product is the DNA section that end carries the polylinker sequence.Use these DNA sections of suitable Restriction Enzyme cracking then, and be connected in the expression vector of using enzymatic lysis, said enzyme can produce and the compatible end of said DNA section.Can buy the synthetic linker that contains a plurality of restriction endonucleases site from a plurality of businessmans.

The expression of above-mentioned antigen-4 fusion protein gene needs subclone in the expression vector with transcription and translation and corresponding operon element, so that recombinant DNA carries out regulatable expression in corresponding host cell.For instance, the system that can be used to realize the fusion rotein high expression level of announcing includes, but are not limited to the pET system (Studier and Moffatt, J.Mol.Biol., 1986,199.) based on T7 polysaccharase and promotor thereof; Trp system (Yanofsky, Nature, 1981,298.) based on the trp promotor; Lac system (Villakamaroff, et al., PNAS, 1978) based on the lac promotor; The tac of mixed t rp and lac promotor, tic, trc system (Brosius et al.J.Biol.Chem, 1985,6.); PBAD system (Guzman et al.J.Bateriol, 1995,177) based on the araBAD promotor; Thermal induction system (Christopher et al., Gene, 1990,87.) based on the PRPL promotor; PQE system (Bujard et al., Methods Enzymol., 1987,155) based on the T5 promotor; And based on (Qing et al., Nat.Biotech, 2004,22.) such as pCold systems of Cold Shock Protein promotor.

As stated, expression system can comprise at least one selective marker.Said mark comprises Tetrahydrofolate dehydrogenase, G418, glutamine synthase or the neomycin resistance as far as eukaryotic cell culture; And the tsiklomitsin, kantlex or the ampicillin resistance gene that are used for intestinal bacteria and other microbial culture.Suitably host's representative example includes but not limited to: bacterial cell, like intestinal bacteria, streptomycete and salmonella typhimurium cell; The fungal cell is like yeast cell (like yeast saccharomyces cerevisiae or pichia pastoris phaff); Insect cell is like fruit bat S2 and noctuid SF9 cell; Zooblast, like CHO, COS, NSO, 293 with the Bowes melanoma cells; And vegetable cell.The appropriate culture medium of above-mentioned host cell and culture condition are known in the art.

Through the transfection of calcium phosphate transfection, the mediation of DEAE-VISOSE, transfection, electroporation, transduction, infection or other method of cation lipid mediation, can nucleic acid of the present invention and nucleic acid recombinant vectors be imported host cell.Said method is described in the laboratory manual of a plurality of standards, like Davis et al., and Basic Methods In Molecular Biology (1986).

The said proteic polynucleotide of the present invention of encoding can be connected in the host, to breed with the carrier that contains selective marker.Generally speaking, can be at throw out, import plasmid vector in the mixture like calcium phosphate precipitation thing or itself and charged lipids.If carrier is a virus, can use suitable packing cell to tie up to and external it packed, transduce to host cell again.

Can identify by successful cell transformed through well-known technology, promptly contain the cell of DNA recombinant vectors of the present invention.For example, the cell that can cultivate importing express recombinant carrier gained is to produce required polypeptide.Collect and lysing cell, use like Southem (1975) J.Mol.Biol.95,503 or Berent et al (1985) Biotech.3,208 described methods detect the existence of DNA in its DNA content.Perhaps, use proteinic existence in the antibody test supernatant.

In case and after the expression system construction success, fibrous root is selected corresponding host cell according to employed expression vector, carries out fermentation culture, most at present carriers all provide controlling element, in the presence of certain inductor, start corresponding proteins and express.

Through well-known method from the reconstitution cell culture, reclaim and purifying said albumen of the present invention comparatively favourable, said method comprise sulfuric acid by or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, dewatering electric charge effect chromatography and lectin chromatography.In some embodiments, can use high performance liquid chromatography (HPLC) to carry out purifying.

In some embodiments, can use above-mentioned one or more chromatography method purifying said albumen of the present invention.In other embodiments; Can use following one or more chromatography column purifying said fusion rotein of the present invention, said chromatography column has: Q sepharose FF post, SP sepharose FF post, Qsepharose High Performance post, Blue sepharose FF post, Blue post, Phenyl SepharoseFF post, DEAE Sepharose FF, Ni-Chelating Sepharose FF post or Methyl post etc.

For separation and purification effectively or secretion target protein, label protein or the label polypeptide (Tag) of being convenient to separation and purification usually also capable of using.Commonly used have glutathione-S-transferase (glutathione S-transferase, GST), six polyhistidyl peptides (His.Tag), a-protein (protein A) and Mierocrystalline cellulose binding site (cellulose binding domain) etc.Through the form of singularity albumen or polypeptide and target protein formation fusion rotein, utilize the special property of described label protein or label polypeptide to separate and purifying after the expression to target protein.Combine with Ni-Chelating Sepharose post specificity like His.Tag.Described label protein or label polypeptide can be removed fusion sequence with the locus specificity protease digestion behind purifying, like available zymoplasm, enteropeptidase and Xa factor etc., to obtain target protein.

In addition, can use the method purifying albumen of describing among the international publication number WO00/44772 (listing this paper in as a reference in full) of the present invention.Those skilled in the art can easily change wherein said method to be used for purifying albumen of the present invention.Can be from comprising that for example the protokaryon or the eucaryon host of bacterium, yeast, higher plant, insect and mammalian cell reclaim albumen of the present invention through the product that recombinant technology produces.

Insulinogenic renaturation

Sphaeroprotein generally has two states: a kind of state of nature for correctly folding, another kind of is the non-correct undernatured state (claiming the sex change attitude again) (Anfinsen, Science, 1973,181) that folds.Term " folds " process that is meant that protein is changed between two states.Term " renaturation " is meant that then protein is returned to this process of state of nature by undernatured state.Term " sex change " is meant that protein is become this process of undernatured state by state of nature.Term " correct folding " is meant that molecular conformation is identical with the state of nature of proinsulin human or Regular Insulin, has correct three pairs of disulfide linkage and other physics and biochemical indicator.Term " non-correct folding " is meant that molecular conformation is different with the state of nature of proinsulin human or Regular Insulin, the mistake pairing of some disulfide linkage possibly occur.

Proteinic sex change and renaturation are the processes of a pair of relative complicacy, and it involves the weak interaction of various complicacies such as hydrogen bond, ionic linkage, Van der Waals force and hydrophobic interaction.Proteinic sex change often results from physics, and the change of electrochemical conditions is such as the rising of temperature and the existence of denaturing agent etc.Term " denaturing agent " is meant and can in the aqueous solution, changes the weak interaction in the protein molecule that cause the material of protein denaturation, denaturing agent commonly used comprises Guanidinium hydrochloride and urea.

Mostly the inclusion body of expressing in the born of the same parents is the aggregate of foreign protein, and it is water insoluble, needs to use double solvents to make its dissolving.In an embodiment preferred, the double solvents of use is selected from Guanidinium hydrochloride and urea.In a preferred embodiment, the double solvents that uses is the 8M urea.

Proinsulin after the redissolution is in denatured state, and in an embodiment preferred, the proinsulin that the present invention is provided as sex change is carried out the method for renaturation.Wherein sex change proinsulin contacts with the denaturing agent of lower concentration at pH 9-pH 11, and preferred denaturing agent is a urea, and preferred concentration is 0.5-2M.Preferred sex change fusion rotein concentration is 0.3-2mg/ml, and more preferred pH is 10.5-11.0.

Mercaptan is for also having-water-soluble mixture of SH group, and it has strong reducing property.The formation of disulfide linkage when mercaptan often forms the certain proportion redox couple with the help protein renaturation with some oxidized form mercaptan dimers.Preferred mercaptan is selected from β mercaptoethanol, WR 34678, second-rate erythritol, reduced glutathion, halfcystine.Preferred oxidized form mercaptan dimer is selected from Sleep-promoting factor B, Gelucystine.In an embodiment preferred, mercaptan and the dimeric ratio of oxidized form mercaptan are 1: 1-1: 10.More be respectively cysteine plus cystine for mercaptan and oxidized form mercaptan dimer in the embodiment preferred at another.Ratio is 1 for the cysteine plus cystine ratio in a highly preferred embodiment: 5-1: 7.

In another embodiment, the correct folding fusion rotein that the present invention provides a kind of proinsulin renaturation to form through above method.

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Embodiment 1, insulin human's preparation

Present embodiment has been described a kind of preparation and has been converted into the purifying proinsulin human and with the proinsulin human

The method of mature human Regular Insulin.The following specifically describes each related step of this process.

A) fermentation: have proinsulin human's expression vector to be transformed in the e. coli bl21 (DE3) clone, the intestinal bacteria that transform are carried out shake-flask culture and high density fermentation cultivation, all can observe expression amount under two kinds of situation up to total protein of cell 20-30%.

B) results inclusion body: the proinsulin human expresses with the inclusion body form in intestinal bacteria; Inclusion body is that what to be not dissolved in water is main aggregate with albumen; The foreign protein of wherein a large amount of enrichment high expression levels, this foreign protein is the proinsulin human in the present embodiment.The cell of results is resuspended at the Tris salt buffer, and use high pressure homogenizer 800bar homogenate twice, centrifugal soluble cell fragment and the foreign protein removed under 12000g.After using twice of Tris/2M urea/1% Triton washing inclusion body, wash once with the Tris salts solution.Gained inclusion body purity is more than 90%.

C) renaturation: above-mentioned inclusion body is dissolved in the 8M urea solution that contains 20mM DTT pH8.0, makes proinsulin concentration at 20-40mg/ml, stirring and dissolving is centrifugal after two hours.Supernatant joins in the pH10.5 solution that contains 3-5mM Gelucystine, 0.3-0.6mM halfcystine of 10 times of volumes 4 ℃ in batches and carries out conventional renaturation.Renaturation time＞48 hour, renaturation yield about 60%.

D) metal ion affinity chromatography: renaturation solution is used through the Ni affinity column with the pH8.0Tris-NaCl solution equilibria, with 5 column volume 400mM imidazoles one-step elutions.

E) trypsinase cracking: above-mentioned elutriant is through the 5KD ultrafiltration and change and be buffered to pH10-11, keeps proinsulin concentration at 8-12mg/ml, adding trypsinase at 1: 1000,37 ℃ cracking 3-4 hour.Cracking process is monitored through HPLC, in case cracking is accomplished, adds phosphoric acid to pH～3.5 termination reactions.

F) reversed phase chromatography: trypsinase cracked proinsulin is through the C18 reversed phase chromatography, to contain the 18%-50% acetonitrile gradient wash-out of 0.16% phosphoric acid.

G) protaminase cracking: the trypsinase split product behind the purifying to pH8.0, adds 1: 1000 protaminase cracking 2 hours through 5KD ultrafiltration exchange buffering, and adding phosphoric acid is to the pH3.5 termination reaction.

H) reversed phase chromatography: protaminase cracked insulin human is through the C18 reversed phase chromatography, to contain the 18%-50% acetonitrile gradient wash-out of 0.16% phosphoric acid.It is the NaCl of 1.0M that the Regular Insulin component is collected back adding final concentration, 0.1M Succinic Acid, pH 6.0-6.4, crystallizing at room temperature, crystallization insulin human product purity＞99%.

I) mass spectroscopy: utilize the MALDI-TOF mass spectrum inspection, measuring purifying human insulin molecule amount is 5807.6, consistent with expection.

Embodiment 2. proinsulin human's expression vector establishments and C polypeptide mutant

The present invention has introduced a kind of proinsulin human's expression system based on the T7 system, and T7 carrier used herein is pCRT7NT (can buy from Invitrogen).

According to insulin human's aminoacid sequence, with the synthetic following section of DNA (SEQ ID NO7) of chemical process, this DNA contains 5 ' end NdeI and 3 ' end EcoRI restriction enzyme site, and coding has histidine-tagged proinsulin human:

catatgcaccaccatcatcaccacggtggccgctttgttaatcagcacctgtgcggctctcacctggtagaggcgctgtatctggtttgtggcgaacgtggcttcttctacactaaaccgacccgtcgcgaagcggaggacctgcaagtgggccaggtcgaactgggtggcggtccgggtgctggttccctgcagccgctggccctggaaggttctctgcagaaacgtggtatcgtggaacagtgctgcacgtctatttgtagcctgtaccagctggaaaactactgcaactaagaattc

With this DNA subclone in the pCRT7NT plasmid, thereby let the proinsulin human be under the regulation and control of T7 promotor and SD sequence.Cloning formed expression system is V3phi (Fig. 2), is used to carry out proinsulin human's expression.It is expressed, and to have histidine-tagged proinsulin human's sequence following:

MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

6 His labels that it had help the insulinogenic collection of renaturation descendant; And GGR had thereafter both guaranteed the abundant exposure of Histidine sequence as link area; The leader sequence that can guarantee again to add can be by the trypsinase cracking, and can not appear in the end product.

And 64 of the LYS of human insulin C-peptide (as above sequence underlined letter section) become Ser, Gly and Val via rite-directed mutagenesis in the present invention, form K64S, K64G and K64V novel C peptide:

Through using by the rite-directed mutagenesis method of the described method improvement of Qiagen QuikChange test kit the K64 of human insulin C-peptide is sported Ser, Gly and Val respectively, the primer is:

K64S forward: GGTTCTCTGCAGTCTCGTGGTATCGTG

Oppositely: CACGATACCACGAGACTGCAGAGAACC

K64G forward: GGTTCTCTGCAGGGCCGTGGTATCGTG

Oppositely: CACGATACCACGGCCCTGCAGAGAACC

K64V forward: GGTTCTCTGCAGGTTCGTGGTATCGTG

Oppositely: CACGATACCACGAACCTGCAGAGAACC

Sudden change back expression product is the proinsulin human who has the novel C peptide:

The K64S proinsulin human:

MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQSRGIVEQCCTSICSLYQLENYCN

The K64G proinsulin human:

MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQGRGIVEQCCTSICSLYQLENYCN

The K64V proinsulin human:

MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQVRGIVEQCCTSICSLYQLENYCN

Result to these three kinds of insulinogenic expression and purifications of novel C peptide shows that the novel C peptide is expressed the proinsulin human does not have influence, and expression product accounts for total bacterial protein more than 20% (Fig. 3).This C peptide has been eliminated Arg (65)-insulin human's by product that wild-type C peptide produces in cracking process, single endonuclease digestion product Regular Insulin corresponding position HPLC is unimodal (Fig. 4) basically.In these three kinds of novel C peptides, be preferably the K64S-C peptide, its expression amount is high (Fig. 3) than other two kinds.Annealing efficiency and enzyme are cut efficient than other two kinds and wild-type all excellent (table 1).

Table 1, wild-type and C polypeptide mutant type proinsulin be each step yield relatively

	Wild-type	K64S	K64G	K64V
					The relative yield of inclusion body	10％	14％	13％	13％
The relative yield of renaturation	55％	60％	55％	55％
					The enzyme cut is to yield	85％	95％	90％	90％

The preparation of embodiment 3 Insulin lispro analogues

It is former and with the former method that is converted into ripe Insulin lispro of Insulin lispro that present embodiment has been described a kind of preparation and purifying Insulin lispro.

Different its Pro28 and the Lys29 of being with the insulin human of Insulin lispro exchange.Form critical sites because the Pro28 position is insulin human's polymer, so the amino-acid substitution in the Insulin lispro makes how this analogue exists with monomeric form.Since Regular Insulin only could bound insulin under monomer acceptor, blood sugar regulation so the main Insulin lispro that exists with monomeric form is rapid-action than regular insulin, is a kind of Semilente Insulin.

Use the former sequence of the Insulin lispro that has the novel C peptide following among the present invention:

MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTKPT RREAED LQVGQVELGGGPGAGSLQPLALEGSLQSRGIVEQCCTSICSLYQLENYCN

In addition; Because the displacement of Pro-Lys forms Lys (28) Pro (29); Pro causes trypsinase can't act on this Lys in the C of Lys end downstream, thereby can further simplify the enzymatic lysis process, is reduced to cracking simultaneously from cracking respectively; Because can also avoid possible deamination in comparatively neutral pH enforcement cracking at high pH.

A) fermentation: depend on the insulinogenic expression vector of dried meat to be transformed in the e. coli bl21 (DE3) clone; The intestinal bacteria that transform are carried out shake-flask culture and high density fermentation cultivation, all can observe expression amount under two kinds of situation up to total protein of cell 20-30%.

B) results inclusion body: Insulin lispro is former expresses with the inclusion body form in intestinal bacteria; Inclusion body is that what to be not dissolved in water is main aggregate with albumen; The foreign protein of wherein a large amount of enrichment high expression levels, this foreign protein is that Insulin lispro is former in the present embodiment.The cell of results is resuspended at the Tris salt buffer, and use high pressure homogenizer 800bar homogenate twice, centrifugal soluble cell fragment and the foreign protein removed under 12000g.After using twice of Tris/2M urea/1% Triton washing inclusion body, wash once with the Tris salts solution.Gained inclusion body purity is more than 90%.

C) renaturation: above-mentioned inclusion body is dissolved in the 8M urea solution that contains 20mM DTT pH8.0, makes the Insulin lispro original content at 20-40mg/ml, stirring and dissolving is centrifugal after two hours.Supernatant joins in the pH10.5 solution that contains 3-5mM Gelucystine, 0.3-0.6mM halfcystine of 10 times of volumes 4 ℃ in batches and carries out conventional renaturation.Renaturation time＞48 hour, renaturation yield about 60%.

E) trypsinase and protaminase copyrolysis: above-mentioned elutriant is through the 5KD ultrafiltration and change and be buffered to pH8.0, keeps the Insulin lispro original content at 8-12mg/ml, to add trypsinase and protaminase, 37C cracking 3-4 hour at 1: 1000.Cracking process is monitored through HPLC, in case cracking is accomplished, adds phosphoric acid to pH～3.5 termination reactions.

F) reversed phase chromatography: protaminase cracked insulin human is through the C18 reversed phase chromatography, to contain the 18%-50% acetonitrile gradient wash-out of 0.16% phosphoric acid.It is the NaCl of 1.0-1.5M that the Insulin lispro component is collected back adding final concentration, 0.1M Succinic Acid, pH 6.0-6.4, crystallizing at room temperature, crystallization Insulin lispro product purity＞99%.

G) mass spectroscopy: utilize the MALDI-TOF mass spectrum inspection, measuring purifying Insulin lispro molecular weight is 5807.6, consistent with expection.

Embodiment 4 biological activity assay

Rabbit with 15 about 2kg of body weight is an experimental subjects, and per 5 rabbit are divided into one group, and totally three groups, wherein first group is experimental group, and administration is the auxilliary insulin product of Ke Xinlai (source embodiment 3); Second group is positive control group, and administration is that gift comes the excellent pleasure of secreting; The 3rd group is negative control group, and administration is a saline water.Gift comes all to be configured to 10IU/ml with the new Insulin lispro of section, according to the pharmacopeia regulation, every rabbit is weighed, and ID is the i.e. 100 μ l/2kg of 1IU/2kg, and negative control group injecting normal saline also is 100 μ l/2kg.Three groups of rabbits are carried out the back subcutaneous injection; The blood sugar concentration test was carried out with blood sugar test paper respectively in 15,30,60,90,150,240 minutes in before the injection and injection back; Experimental result shows, the new Insulin lispro product of section (source embodiment 3) is excellent with gift, and to secrete happy biological activity consistent.(Fig. 5)

Scope of the present invention does not receive the restriction of said specific embodiments, and said embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating all respects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to the description and the accompanying drawing of preceding text.Said improvement also falls within the scope of appended claims.Every piece of reference that preceding text are mentioned is listed this paper in as a reference all in full.

Claims

1. proinsulin human's fusion rotein; Hold to C end from N and to form by four sections peptide chains of X-B-C-A; Wherein said X peptide chain is not for having or the guiding peptide; Said B-C-A peptide chain for its aminoacid sequence shown in SEQ IDNO.3 or for have the two mutants of hypoglycemic activity at least with aminoacid sequence 90% homology shown in the SEQ ID NO.3 and its ripe body, it is characterized in that Lys 64 residues of said C peptide C end are replaced by non-basic aminoacids.

2. according to the said proinsulin human's fusion rotein of claim 1, it is characterized in that said non-basic aminoacids is one of among Ser, Gly or the Val.

3. according to the said proinsulin human's fusion rotein of claim 1, the aminoacid sequence that it is characterized in that said C peptide is shown in SEQ ID NO.4.

4. according to the said proinsulin human's fusion rotein of claim 1, the aminoacid sequence that it is characterized in that said C peptide is shown in SEQ ID NO.5.

5. according to the said proinsulin human's fusion rotein of claim 1, the aminoacid sequence that it is characterized in that said C peptide is shown in SEQ ID NO.6.

6. according to the said proinsulin human's fusion rotein of claim 1, it is characterized in that said guiding peptide holds the C end to comprise from N: the first peptide section follows 6-10 Histidine closely by methionine(Met); The second peptide section is that 1-3 glycocoll-Serine repeats to follow closely Methionin or l-arginine.

7. according to the said proinsulin human's fusion rotein of claim 6, it is characterized in that the said first peptide section aminoacid sequence is shown in SEQ ID NO.1.

8. according to the said proinsulin human's fusion rotein of claim 6, it is characterized in that the said second peptide section aminoacid sequence is shown in SEQ ID NO.2.

9. according to the said proinsulin human's fusion rotein of claim 1, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

10. according to the said proinsulin human's fusion rotein of claim 1, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

11. according to the said proinsulin human's fusion rotein of claim 1, the Argine Monohydrochloride sequence that it is characterized in that said proinsulin human's fusion is shown in SEQ ID NO.16.

12. the nucleotide sequence of the said proinsulin human's fusion rotein of the claim 1 of encoding.

13. according to the said nucleotide sequence of claim 12, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

14. according to the said nucleotide sequence of claim 12, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

15. according to the said nucleotide sequence of claim 12, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

16. recombinant vectors that contains the nucleotide sequence of the said proinsulin human's fusion rotein of coding claim 1.

17. according to the said recombinant vectors of claim 16, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

18. according to the said recombinant vectors of claim 16, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

19. according to the said recombinant vectors of claim 16, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

20., it is characterized in that said recombinant vectors is the pCRT7NT plasmid according to each said recombinant vectors of claim 16～19.

21. transformant that contains the recombinant vectors of the said proinsulin human's fusion rotein of coding claim 1 nucleotide sequence.

22. according to the said transformant of claim 21, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.14.

23. according to the said transformant of claim 21, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.15.

24. according to the said transformant of claim 21, the aminoacid sequence that it is characterized in that said proinsulin human's fusion rotein is shown in SEQ ID NO.16.

25., it is characterized in that said transformant body is intestinal bacteria according to the said transformant of claim 21.

26. an insulin human preparation method comprises the following steps:

A) make up the said recombinant vectors of claim 16;

B) obtain transformant;

C) fermentation;

D) results contain the tunning part of proinsulin human's fusion rotein;

E) renaturation;

F) purifying protein;

G) trypsinase cracking: through the 5KD ultrafiltration, pH10～11 keep purifying protein concentration at 8～12mg/ml, adding trypsinase at 1: 1000,37 ℃ cracking 3-4 hour, add phosphoric acid to pH～3.5 termination reactions, remove trypsinase;

H) protaminase cracking: through the 5KD ultrafiltration, pH8.0 adds 1: 1000 protaminase cracking 2 hours, adds phosphoric acid to the pH3.5 termination reaction, removes protaminase;

I) albumen recrystallization promptly obtains the insulin human.

27. preparation method according to claim 26, it is characterized in that renaturation among the said step e be sex change proinsulin human's fusion rotein with contain mercaptan, oxidized form mercaptan dimer contacts with denaturant solution.

28. preparation method according to claim 26 is characterized in that said proinsulin human's fusion rotein concentration is 0.3～2mg/ml.

29. preparation method according to claim 27 is characterized in that said denaturing agent is that urea is or/and Guanidinium hydrochloride.

30. preparation method according to claim 27 is characterized in that said denaturing agent is a urea.

31. preparation method according to claim 30 is characterized in that said urea concentration is 0.5～1.5M.

32. preparation method according to claim 27, the pH scope that it is characterized in that said renaturation is 9～11.

33. preparation method according to claim 27, the pH scope that it is characterized in that said renaturation is 10.5～11.0.

34. preparation method according to claim 27 is characterized in that said mercaptan and the dimeric ratio of oxidized form mercaptan are 1: 1～1: 10.

35. preparation method according to claim 27 is characterized in that said mercaptan is halfcystine, oxidized form mercaptan dimer is a Gelucystine.

36. preparation method according to claim 35, the ratio that it is characterized in that said halfcystine and Gelucystine is 1: 5～1: 7.