CN101235084A

CN101235084A - Method for preparing bone morphogenic protein BMP-2 mature peptide

Info

Publication number: CN101235084A
Application number: CNA2007100671383A
Authority: CN
Inventors: 林陈水; 付水星; 任慧颖; 黎小军; 徐伟; 于真真; 许明; 郑小玲; 钱俊青
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2007-01-31
Filing date: 2007-01-31
Publication date: 2008-08-06

Abstract

The invention provides a preparation method of recombination bone morphogenetic protein BMP-2 mature peptide, comprising deleting the 2-18 amino acid encoding genes of DsbA gene signal peptide, keeping the codon of the first methilanin as starting codon to obtain a section recombined in expression carrier to obtain recombination plasmid 1, recombining the DNA of the bone morphogenetic protein BMP-2 complete mature peptide in the descending of the DsbA gene of recombination plasmid 1 to obtain recombination plasmid 2, transferring the recombination plasmid 2 into the thallus of expression host strain to obtain engineering bacteria, via cultivation and adding inducer to express target gene, breaking bacteria, separating and purifying to obtain DsbA-BMP-2 fusion protein, cutting the DsbA-BMP-2 fusion protein via prolease, separating to obtain the one morphogenetic protein BMP-2 mature peptide. The inventive product has same amino acid sequence of natural BMP-2 mature peptide, to realize soluble cell fusion expression, while the fusion protein can be purified further via Ni2+ affinity chromatography to simplify the purification before BMP-2 renaturation.

Description

A kind of method for preparing bone morphogenic protein BMP-2-2 mature peptide

(1) technical field

The present invention relates to prepare the method for bone morphogenic protein BMP-2-2 mature peptide, particularly obtain method with the on all four BMP-2 mature peptide of natural B MP-2 mature peptide aminoacid sequence.

(2) background technology

(Bone Morphogenetic Protein-2 BMP-2) belongs to the transforming growth factor superfamily member to bone morphogenesis protein-2.BMP has the undifferentiated mesenchymal cell of inducing to cartilage, bone differentiation, and the formation and the growth of embryo's bone and multiple organ played important regulating effect.The induced osteogenesis activity of BMP uniqueness makes it in skeletal injury and illness, as the fracture, bone is damaged and the oral cavity quite has broad application prospects in the face surgery.In recent years, by the DNA recombinant technology, the reorganization of kind more than ten bmp protein has all obtained broad research in basis and clinical application, and in organic evolution, the research of aspect effects such as growth and heredity just deepens continuously to BMP.

BMP first mode with precursor in cell produces, and comprises the mature peptide of signal peptide, propetide and carboxyl terminal.The halfcystine that contains 7 high conservatives in the mature peptide, they form three intrachain disulfide bonds and interchain disulfide bond and two polypeptide chains are connected to dimer, the BMP precursor protein is through N position glycosylation, proteolytic enzyme cleaves and Dimerized and form the BMP mature peptide, its active formation mostly is its homodimer, does not have glycosylated homodimer still to have biologic activity.Multiple histocyte is in the different steps expressed BMP of fetal development, and isolated BMP from multiple animal bone tissues such as ox, mouse, rabbit, people.In addition, all contain abundant BMP in the tissues such as dentine, placenta, osteosarcoma.The application of radiation immunological technique can detect the BMP in the serum, and the about 14-18n/mlg of grownup, children are 20-72ng/ml.

First amino acid of natural B MP-2 mature peptide is not methionine(Met), conventional technology of preparing is the N-terminal interpolation methionine(Met) at mature peptide or truncation type, utilize intestinal bacteria directly to express, the inclusion body renaturation method prepares BMP-2, rhBMP-2 as East China gene engineering institute exploitation truncation type, after removing 7 amino acid of N end, methionine(Met) of artificial interpolation, with its codon ATG as initiator codon, efficiently express in Escherichia coli cell, the inclusion body protein renaturation has obtained the biological activity homodimer.

Before the present invention, " preparation of human bone morphogenesis protein-2 eukaryon expression plasmid, transfection and expression " that modern medicine 33 volume 5 phases (2005/10) Kang Chunyu Zhang Qingguo deliver introduced the method for preparing the total length eukaryon expression plasmid pcDNA3.1-BMP2 that carries hBMP-2, by liposome method with the transfection of resulting reorganization eucaryon plasmid in the rabbit bone marrow stroma cell, carry out gradient screening with G418, thus obtain can stably express hBMP-2 cell clone.Adopt RT-polymerase chain reaction (RT-PCR) to detect the expression of marrow stromal cell (MSCs) hBMP-2mRNA.

Chinese patent application CN1484651 has introduced protein has been expressed in prokaryotic organism, this is made up of a kind of proteinic presequence or its part of TGF-beta superfamily by the marking protein amino terminal, protein or the proteinic ripe zone of another kind of TGF-beta superfamily are connected on this protein, and the protein that is connected has the homology with maturation zone BMP-2 at least 35%.Protein expression is having at least a part of protein to obtain with the form of inclusion body.

As everyone knows, it is with low cost to utilize protokaryon system expression pharmaceutical protein to have, and is easy to the advantage of scale operation.Because deglycosylated BMP-2 still has biologic activity, this has created condition for protokaryon system expressed BMP-2, the recombinant human B MP-2 that gene engineering institute in East China successfully utilizes intestinal bacteria to prepare to have biologic activity.But product that obtains and natural BMP-2 aminoacid sequence are inconsistent, are used for human body and may produce immunogenicity.Have the low advantage of cost though utilize inclusion body to prepare target protein, contain certain foreign protein and nucleic acid in the inclusion body, target protein accounts for about 80% of total protein.

(3) summary of the invention

The invention provides a kind of method for preparing bone morphogenic protein BMP-2-2 mature peptide, the product that this method makes can be in full accord with natural B MP-2 mature peptide aminoacid sequence, for realizing purpose of the present invention, the present invention adopts following technical scheme, and the method that the present invention prepares the BMP-2 mature peptide comprises following sequential steps:

(1) the 2nd～18 amino acid encoding gene of deletion DsbA gene signal peptide, the codon that keeps first methionine(Met) is as initiator codon, obtain the encoding dna fragmentation of the DsbA that do not contain signal peptide is reconstituted in this fragment in the expression vector, obtains recombinant plasmid 1; Described DsbA gene can escherichia coli chromosome DNA come out as template amplification, also the plasmid that contains the DsbA gene such as available pET comes out as template amplification, it is easier to do template with plasmid.Also can manually obtain purpose DsbA gene theoretically with the method for chemosynthesis.

(2) will the encode DNA of the complete mature peptide of bone morphogenic protein BMP-2-2 is reconstituted in the downstream of DsbA gene in the recombinant plasmid 1, obtains recombinant plasmid 2;

(3) recombinant plasmid 2 is transformed in the thalline of host strain, obtains engineering bacteria, described host bacterium is the DE3 lysogenic bacteria of the escherichia coli host of no thioredoxin reductase defective;

(4) engineering bacteria is cultivated in containing antibiotic substratum, adds inductor and makes destination gene expression;

(5) the engineering bacteria cell is through broken bacterium, and separation and purification obtains the BMP-2 fusion rotein;

The proteolytic enzyme enzyme is cut the BMP-2 fusion rotein, obtains described bone morphogenic protein BMP-2-2 mature peptide through separation.

Wherein, the plasmid that contains the DsbA gene in the described step (1) is preferably pET39.Also contain this gene among the bacillus coli gene group DNA, can obtain the gene of encoding D sbA with the PCR method amplification.Host strain is preferably e. coli bl21 (DE3) in the described step (3).Being expressed under the shortage oxidation envrionment conditions of goal gene carried out in the described step (4).Proteolytic enzyme is enteropeptidase or recombinant enterokinase light chain in the described step (6).

The preparation method of described bone morphogenic protein BMP-2-2 mature peptide, in the described step (2), between the DNA of the complete mature peptide of bone morphogenic protein BMP-2 in the recombinant plasmid 2-2 and the downstream of DsbA gene, also reorganization has the sequence of coding " Linker-6His-proteolytic enzyme recognition site ".Described 6His parts also can be positioned at the upstream of DsbA gene, and the proteolytic enzyme recognition site is an enteropeptidase.Specifically, for example, get the plasmid pET39 that contains the DsbA gene, the DsbA signal peptide gene among the deletion pET39 obtains recombinant plasmid 1:pET39 (SP-); To the encode DNA splicing of dna fragmentation with coding bone morphogenic protein BMP-2-2 mature peptide of " Linker-6His-Eksite " obtains the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 "; The dna fragmentation of " Linker-6His-Eksite-BMP-2 " will encode again by the downstream that restriction enzyme BspTI and BamHI are reconstituted in DsbA gene among the recombinant plasmid 1:pET39 (SP-), obtain recombinant plasmid 2:pET39 (SP-BMP-2).

Described method recommends in the following order step to carry out:

(1) with the plasmid pET39 that contains the DsbA gene as pcr template, utilize the 2nd～18 the amino acid encoding gene of DsbA signal peptide gene among the round pcr deletion pET39, the codon that keeps first methionine(Met) is as initiator codon, the dna fragmentation that obtains is reconstituted in the expression vector, obtains recombinant plasmid 1:pET39 (SP-);

(2) DNA of the complete mature peptide of bone morphogenic protein BMP-2-2 is reconstituted in the downstream of DsbA gene in the recombinant plasmid 1, obtains recombinant plasmid 2:pET39 (SP-BMP-2);

(3) recombinant plasmid 2:pET39 (SP-BMP-2) is transformed in the colibacillary thalline of host strain, obtains engineering bacteria;

(4) engineering bacteria is cultivated in the Luria-Bertani substratum, makes Target Fusion albumen obtain expressing;

(6) cut the BMP-2 fusion rotein with the enteropeptidase enzyme, obtain described bone morphogenic protein BMP-2-2 mature peptide through separation.

Further, the preparation method of bone morphogenic protein BMP-2-2 mature peptide, preferably step is carried out in the following order:

(1) be template with the plasmid pET39 that contains the DsbA gene, delete the 2nd～18 the amino acid encoding gene of DsbA signal peptide gene in this plasmid, the codon that keeps first methionine(Met) is as initiator codon, the fragment that obtains is reconstituted in the expression vector, obtains recombinant plasmid 1:pET39 (SP-);

(2) will the encode DNA splicing of dna fragmentation with coding bone morphogenic protein BMP-2-2 mature peptide of " Linker-6His-Eksite " obtains the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 "; The dna fragmentation of " Linker-6His-Eksite-BMP-2 " of will encoding again is reconstituted in the downstream of DsbA gene among the recombinant plasmid 1:pET39 (SP-), obtains recombinant plasmid 2:pET39 (SP-BMP-2);

(3) recombinant plasmid 2:pET39 (SP-BMP-2) is transformed in the thalline of host strain E.coli BL21 (DE3), obtains engineering bacteria;

(4) engineering bacteria is in the Luria-Bertani substratum, and 30 ℃, 200r/min are cultivated OD ₆₀₀About 0.5 o'clock, adding final concentration was 0.1～1mM inductor isopropylthio-, and 30～37 ℃ are continued to cultivate 2～10h down, make destination gene expression;

(5) after the fermentation ends, centrifugal collection engineering bacteria cell, centrifugal behind the broken bacterium, the collection supernatant liquor is crossed and is contained Ni ²⁺Chelate column, with the pre-wash-out of 50mM imidazole buffer, containing the buffer solution elution target protein of imidazoles 120～150mM, collect elutriant; 50mM imidazole buffer (pH7.3) preparation: get imidazoles 0.68g and sodium-chlor 1.17g, add water and make and be dissolved into 100ml, add 0.1mol/ml hydrochloric acid soln 42.2ml, thin up is to 200ml, promptly again.

(6) in the elutriant that contains the BMP-2 fusion rotein, add the diethylamine tetraacethyl of final concentration 0.1～1mM, and enteropeptidase 5U/mL, carry out enzyme under 25 ℃ to cut, the proteolytic enzyme enzyme is cut fusion rotein, obtains described bone morphogenic protein BMP-2-2 mature peptide through separation.U is the enzyme unit that lives, and is defined as under 23 ℃ of conditions, 50 μ g trypsinogens 95% is converted into the required enzyme amount of trypsinase in 8 hours, down with.

Further again, specifically, the preparation method of bone morphogenic protein BMP-2-2 mature peptide step in the following order carries out:

(1) be masterplate with the plasmid pET39 that contains the DsbA gene,

Primer: DsbA-1:5 '-AAGCATATGGCGCAGTATGAAGAT-3 ',

DsbA-2：5’-TCGCTTAAGTATTTCACTGT-3’，

PCR parameter: 94 ℃ of 3min

3 circulations of 72 ℃ of 1min of 40 ℃ of 30s of 94 ℃ of 30s

27 circulations of 72 ℃ of 1min of 48 ℃ of 30s of 94 ℃ of 30s

72℃?10min

4℃ 10min

Cut PCR product and pET39 plasmid with NdeI and BspTI enzyme, make original DsbA gene among the dna fragmentation displacement pET39 that does not contain signal peptide gene, obtain not contain the recombinant plasmid 1:pET39 (SP-) of DsbA signal peptide gene;

(2) will the encode dna fragmentation of " Linker-6His-Eksite ":

ATA CTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCATCATCATGATGACGATGACA AA, wherein CTTAAGBe the BspTI recognition sequence,, obtain the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 " with the DNA splicing of coding bone morphogenic protein BMP-2-2 mature peptide:

Primer Linker-1:5 '-ATA CTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCAT-3 '

Primer Linker-2:5 '-TTGCTTATGCTTTGCTTGTTTGTCATCGTCATCATGATGATGATGATGATGACC 3 '

Primer BMP2-1:5 '-GACGATGACAAA CAAGCAAAGCATAAGCAA-3 '

Primer BMP2-2:5 '-TTCGGATCCTTAGCGACAGCCACAACCTT-3 '

Getting concentration is primer Linker-1 and each 2uL of Link-2 of 10uM, adds 2uL dNTP, and 5uL10 * DNA Polymerase Buffer mends ddH ₂O to 50uL reaction system, boiling water bath 10min puts into 50 ℃ of water-bath cooling 3min, adds 2.5U Pfu DNA polymerase, changes 72 water-bath 10min over to, makes two primers template each other, forms the dna double chain, obtains reaction product A;

As forward and reverse primer, is that template carry out pcr amplification with the BMP-2 gene with primer BMP2-1 and BMP2-2, and annealing temperature is made as 50 ℃, extends time 30s, obtains reaction product B;

As pcr template, carry out gene splicing with primer Linker-1 and BMP2-2 as forward and reverse primer with product A and product B, obtain the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 ", reaction system and reaction parameter are as follows:

PCR parameter: 94 ℃ of 3min

30 circulations of 72 ℃ of 1min of 60 ℃ of 30s of 94 ℃ of 30s

72℃?10min

4℃ 10min；

(3) will the encode dna fragmentation of " Linker-6His-Eksite-BMP-2 " is reconstituted in the downstream of DsbA gene among the recombinant plasmid pET39 (SP-), obtains recombinant plasmid 2:pET39 (SP-BMP-2);

(4) recombinant plasmid 2:pET39 (SP-BMP-2) is transformed in the thalline of host strain E.coli BL21 (DE3), obtains engineering bacteria;

(5) engineering bacteria gets engineering bacteria seed liquid through seed culture in the Luria-Bertani substratum, cultured engineering bacteria seed liquid is inoculated into by 1% inoculum size that to shake bottled liquid measure be 20% Luria-Bertani substratum fermentation culture, and 30 ℃, 200r/min are cultivated OD ₆₀₀Be about at 0.5 o'clock adding inductor isopropylthio-to final concentration is 0.2mM, continues under 30 ℃ of temperature to cultivate 3h, makes destination gene expression;

(6) after the fermentation ends, centrifugal collection engineering bacteria cell, centrifugal after the carrying out ultrasonic bacteria breaking, the collection supernatant liquor is crossed and is contained Ni ²⁺Chelate column, with the pre-wash-out of 50mM imidazole buffer,, collect elutriant with Tris-HCl (pH7.8) buffer solution elution that contains the 150mM imidazoles;

(7) in the elutriant that contains the BMP-2 fusion rotein, add the diethylamine tetraacethyl of final concentration 1mM, and enteropeptidase 5U/mL, carry out enzyme under 25 ℃ to cut, the proteolytic enzyme enzyme is cut fusion rotein,

Obtain described bone morphogenic protein BMP-2-2 mature peptide through separation.

The present invention at first will be the removal of DsbA signal peptide gene, to express in the born of the same parents that realize DsbA, disulfide linkage forms albumin A (disulfide bond formation protein A, DsbA) be a kind of disulfide bond isomerase that the auxiliary multiple protein that contains disulfide linkage correctly folds and has biologic activity in the colibacillus periplasm cell. found by Bardwell in 1991, be first found protein in this family, it is present in the E.coli periplasmic space (periplasm), has the strongest oxidisability, molecular weight is that 21kD.DsbA can form disulfide linkage by the interior protein of catalysis periplasmic space, and has good solvability.Natural DsbA gene contains signal peptide gene, instructs the DsbA secretion to the periplasmic space with well-oxygenated environment.The BMP-2 fusion rotein that contains seven cysteine residues forms polymer in the periplasmic space expression meeting with well-oxygenated environment by disulfide linkage.For the DsbA fusion rotein is expressed in the born of the same parents that lack well-oxygenated environment, must delete the dna fragmentation of its coded signal peptide 2-18 amino-acid residue, keep first amino acids methionine codon ATG as its beginning codon of translation.

Design 5 ' mutant primer, with the pET39 that carries the DsbA gene is template, utilize the round pcr amplification not contained the dna fragmentation of the encoding D sbA of signal peptide gene, DNA is reconstituted among the pET39 after utilizing restriction enzyme NdeI and BspTI to suddenly change, the engineering bacteria that obtains plasmid pET39 (SP-) structure coding " DsbA-Linker-6His Eksite-BMP-2 " fusion rotein is spatially separate for two major portions that make fusion rotein, designs GSGSG as connection peptides (G, S are respectively the single-letter abbreviations of glycine, Serine) at the C of DsbA end.The separation and purification of fusion rotein for convenience is in the C of Linker end design 6His affinity tag, for expression product can be used Ni ²⁺The affinity chromatography separation and purification creates conditions.In order to obtain and the on all four product of natural B MP-2 aminoacid sequence, at the recognition site (DDDDK) (D, K are respectively the single-letter abbreviations of aspartic acid, Methionin) of the N of BMP-2 end design enteropeptidase.Because the laboratory has had the gene of encoding BMP-2, in order to make up this project bacterium, on the basis of deleting the DsbA signal peptide gene, the BspTI site that utilizes DsbA gene 3 ' end to contain, the DNA of design coding " Linker-6His-Eksite " and the gene splicing of encoding BMP-2, DNA is reconstituted in pET39 (among the SP (-) to utilize restriction enzyme BspTI and BamHI will suddenly change afterwards.Recombinant plasmid transformed expressive host E.coli BL21 (DE3) obtains engineering bacteria.

The expression of engineering bacteria and the separation and purification of expression product, engineering bacteria grows to OD in containing antibiotic LB substratum ₆₀₀About=0.5, adding final concentration is the IPTG of 0.1～1mM, makes destination gene expression, and 12000rpm after carrying out ultrasonic bacteria breaking is centrifugal for the engineering bacteria cell, utilizes SDS-PAGE to analyze precipitation and supernatant, determines the solvability of fusion rotein.

Soluble fusion rotein is through Ni ²⁺Affinity chromatography one step separation and purification can obtain purity greater than 95% BMP-2 fusion rotein.

The monomeric acquisition of BMP-2 mature peptide, cut fusion rotein with the recombinant enterokinase enzyme, because the solubleness of BMP-2 mature peptide in the aqueous solution is very little, cut at enzyme and to occur deposited phenomenon in the process, after the centrifugation, DsbA carrier and enteropeptidase are in supernatant liquor, and most of BMP-2 mature peptide monomer is in precipitation, thereby successfully obtain BMP-2 mature peptide monomer, BMP-2 mature peptide monomer can be used for the dimer that the renaturation method preparation has biologic activity.

Utilize the inventive method, can obtain and the on all four BMP-2 mature peptide of natural B MP-2 aminoacid sequence.And adopt after the amalgamation and expression strategy, the fusion rotein solvability is good, contains the label of affinity chromatography, can use Ni ²⁺The method separation and purification fusion rotein of affinity purification, purity can reach more than 95%.Enzyme is cut back BMP-2 mature peptide monomer and is precipitated out owing to the solubleness in the aqueous solution is little, can be with simple centrifugation target protein and fusion partner (DsbA carrier albumen).

The present invention compared with prior art, its beneficial effect is embodied in: in full accord with product and natural B MP-2 mature peptide aminoacid sequence that the inventive method makes; Amalgamation and expression is realized solubility expression, and fusion rotein can be used Ni ²⁺The affinity chromatography single step purification has further been simplified the preceding purification step of BMP-2 renaturation.

(4) description of drawings

Fig. 1 is the plasmid map of recombinant plasmid pETDsbA-BMP.

(5) embodiment:

Below with specific embodiment technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto:

The removal of embodiment 1:DsbA signal peptide gene

Design of primers: DsbA-1:5 ' AAGCATATGGCGCAGTATGAAGAT 3 ' 24bp

DsbA-2：5’TCGCTTAAGTATTTCACTGT 3’20bp

Pcr template: pET39 plasmid

PCR parameter: 94 ℃ of 3min

3 circulations of 72 ℃ of 1min of 40 ℃ of 30s of 94 ℃ of 30s

27 circulations of 72 ℃ of 1min of 48 ℃ of 30s of 94 ℃ of 30s

72℃?10min

4℃ 10min

Cut PCR product and pET39 plasmid with NdeI and BspTI enzyme, make original DsbA gene among the dna fragmentation displacement pET39 that does not contain signal peptide gene, the recombinant plasmid called after pET39 (SP-) of acquisition.

Embodiment 2: the acquisition of coding " Linker-6His-Eksite-BMP-2 " gene

At first according to the dna fragmentation of intestinal bacteria preference codon design coding " Linker-6His-Eksite ", sequence is as follows: ATA CTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCATCATCATGATGACGATGACA AA, wherein line part is the BspTI recognition sequence.For this dna fragmentation and BMP-2 gene are spliced smoothly, design following four primers, splice with the SOE technology.

Primer Linker-1:

5’ATA CTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCAT?3’46bp

Primer Linker-2:

5’TTGCTTATGCTTTGCTTGTTTGTCATCGTCATCATGATGATGATGATGATGACC?3’54bp

Primer BMP2-1:

5’GACGATGACAAA CAAGCAAAGCATAAGCAA?3’?30bp

Primer BMP2-2:

5’TTCGGATCCTTAGCGACAGCCACAACCTT 3’?30bp

Working method:

Because primer Linker-1 and primer Linker-2 have the 18bp reverse complemental, getting concentration is primer Linker-1 and each 2uL of Link-2 of 10uM, add 2uL dNTP, 5uL 10X DNA PolymeraseBuffer mends ddH2O to 50uL reaction system, boiling water bath 10min, put into 50 ℃ of water-bath cooling 3min, add 2.5U Pfu DNA polymerase, change 72 water-bath 10min over to, make two primers template each other, form the dna double chain.Reaction product called after product A.

Again the amplification of BMP-2 gene: as forward and reverse primer, the BMP-2 gene that has obtained with the laboratory is that template is carried out pcr amplification with primer BMP2-1 and BMP2-2, and annealing temperature is made as 50 ℃, extends time 30s, the product called after product B that obtains.

Gene splicing: as pcr template, carry out gene splicing with primer Linker-1 and BMP2-2 as forward and reverse primer with product A and product B, reaction system and reaction parameter are as follows:

PCR parameter: 94 ℃ of 3min

30 circulations of 72 ℃ of 1min of 60 ℃ of 30s of 94 ℃ of 30s

72℃?10min

4℃ 10min

The PCR product called after product C that obtains is the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 ".

Embodiment 3: engineering bacteria makes up

Cut product C with BspTI and BamHI enzyme, Protocols in Molecular Biology is reconstituted among the pET39 (SP-) routinely, obtain recombinant plasmid pETDsbA-BMP (plasmid map is seen Fig. 1), recombinant plasmid pETDsbA-BMP Transformed E .coli BL21 (DE3) preserves this project bacterium after order-checking is identified.Be used for expression study and production.

Embodiment 4: express test in the born of the same parents of " DsbA-Linker-6His-Eksite-BMP-2 " fusion rotein

In the 3mL LB substratum that contains the 50ug/mL kantlex, 30 ℃, 200r/min were cultivated 16 hours with single bacterium colony of aseptic toothpick picking positive colony, engineering bacteria seed liquid.

Cultured engineering bacteria seed liquid is inoculated into by 1% inoculum size that to shake bottled liquid measure be in 20% the Luria-Bertani substratum, and 30 ℃, 200r/min are cultivated OD ₆₀₀About 0.5 o'clock, adding inductor IPTG was 0.2mM to final concentration, and induction exogenous gene is expressed, and continued to cultivate 3h under 30 ℃ of temperature.Collect thalline and do full bacterium SDS-PAGE analysis, can obtain the expression product that apparent molecular weight is 36kD.

Embodiment 5: the solvability analysis and the separation and purification of fusion rotein

After the fermentation ends, with the centrifugal collection engineering bacteria of 3000rpm rotating speed cell, with the resuspended thalline of 20mM Tris-HClpH8.0 damping fluid, after the carrying out ultrasonic bacteria breaking, centrifugal with centrifugal 12000rpm rotating speed, collecting precipitation and last asking are precipitated with 4M Urea respectively, the dissolving of 0.1M NaCl solution, analyze the content of precipitation and supernatant target protein with SDS-PAGE, among the present invention, fusion rotein mainly is present in the supernatant liquor, illustrates that fusion rotein is present in the engineering bacteria cell with soluble form.

(1) separation and purification of fusion rotein

Because fusion rotein contains 6His affinity purification label, fusion rotein can be adsorbed in Ni specifically ²⁺On the chelate column,,, collect elutriant with Tris-HCl, the pH7.8 buffer solution elution target protein that contains imidazoles 150mM with the pre-wash-out of 50mM imidazole buffer;

(2) enzyme of fusion rotein is cut and the monomeric acquisition of BMP-2 mature peptide

At Ni ²⁺In the elutriant that contains fusion rotein of chelating affinity chromatography, adding final concentration is the EDTA of 1mM and the enteropeptidase of 5U/mL, carrying out enzyme under 25 ℃ of conditions cuts, cut at enzyme and to discharge BMP-2 mature peptide monomer in the process, because the monomeric solvability of BMP-2 mature peptide is very little, enzyme cuts that solution gradually becomes muddy by clarification in the process.

After enzyme is cut end, with rotating speed centrifugal collecting precipitation greater than 5000rpm, with 20mM Tris-HCl washing precipitation once, be precipitated as purity greater than 90% BMP-2 mature peptide monomer, this monomer can be used for further renaturation has biologic activity with preparation reorganization homodimer.

15 determined amino acid sequence results of the monomeric N-end of BMP-2 mature peptide are N-Gln Ala Lys HisLys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys, and are in full accord with natural BMP-2 mature peptide n terminal amino acid sequence.

Embodiment 6: obtain homodimer with the external renaturation of the BMP-2 mature peptide monomer that obtains

Damping fluid dissolving BMP-2 monomer precipitation with the Tris-HCl pH8.5 that contains 8M urea, 10mM dithiothreitol (DTT) (DDT), 5mM EDTA, the room temperature lower magnetic force stirred 2 hours, with the dilution method renaturation, the final concentration of renaturation system essential substance is as follows: 0.5M L-arginine, 0.1M sodium-chlor, 0.5M urea, 1mM reduced glutathion GSH, 0.1mM Sleep-promoting factor B GSSG.Place more than 24 hours at 4-10 ℃ of refrigerator, analyze dimeric formation situation with irreducibility SDS-PAGE, along with the prolongation of renaturation time, apparent molecular weight is that the protein of 25kD increases gradually.This band disappears when analyzing with reductibility SDS-PAGE, can infer that apparent molecular weight is that the protein of 25kD is the dimer of BMP-2 mature peptide.

Embodiment 7: the gene sequencing analysis

After testing, the gene order of gained BMP-2 fusion rotein of the present invention following (the line part is a BMP-2 mature peptide gene):

ATGGCGCAGTATGAAGATGGTAAACAGTACACTACCCTGGAAAAACCGGTAGCTGGCGCGCCGCAAGTGCTGGAGTTTTTCTCTTTCTTCTGCCCGCACTGCTATCAGTTTGAAGAAGTTCTGCATATTTCTGATAATGTGAAGAAAAAACTGCCGGAAGGCGTGAAGATGACTAAATACCACGTCAACTTCATGGGTGGTGACCTGGGCAAAGATCTGACTCAGGCATGGGCTGTGGCGATGGCGCTGGGCGTGGAAGACAAAGTGACTGTTCCGCTGTTTGAAGGCGTACAGAAAACCCAGACCATTCGTTCTGCTTCTGATATCCGCGATGTATTTATCAACGCAGGTATTAAAGGTGAAGAGTACGACGCGACGTGGAACAGCTTCGTGGTGAAATCTCTGGTCGCTCAGCAGGAAAAAGCTGCAGCTGACGTGCAATTGCGTGGCGTTCCGGCGATGTTTGTTAACGGTAAATATCAGCTGAATCCGCAGGGTATGGATACCAGCAATATGTTTGTTCAGCAGTATGCTGATACAGTGAAATACTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCATCATCATGATGACGATGACAAA CAAGCAAAGCATAAGCAACGGAAG AGGCTGAAATCCTCCTGCAAAAGACATCCGCTGTATGTGGATTTTAGCGA TGTGGGCTGGAACGATTGGATTGTGGCGCCGCCGGGCTATCATGCGTTTT ATTGCCATGGCGAATGCCCGTTTCCGCTGGCGGATCATCTGAACTCCACC AACCATGCGATTGTGCAGACCCTGGTGAACTCTGTGAACAGCAAAATTC CGAAAGCGTGCTGTGTGCCGACCGAACTGAGCGCGATTTCGATGCTGTA TCTGGATGAAAACGAAAAAGTGGTGCTGAAAAACTATCAGGATATGGTG GTGGAAGGTTGTGGCTGTCGCTAA；

The aminoacid sequence of gained BMP-2 fusion rotein of the present invention following (the line part is a BMP-2 mature peptide amino acid):

MAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIKGEEYDATWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMFVQQYADTVKYLSEKKGSGSGHHHHHHDDDDK QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAF YCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGCGCR*；

Conclusion: in full accord with bone morphogenic protein BMP-2-2 mature peptide and natural B MP-2 mature peptide aminoacid sequence that the inventive method makes.

Sequence table .ST25

SEQUENCE?LISTING

＜110〉Zhejiang Polytechnical University

＜120〉a kind of method for preparing bone morphogenic protein BMP-2-2 mature peptide

<130>

<160>8

<170>PatentIn?version?3.2

<210>1

<211>65

<212>DNA

<213>Unknown

<220>

＜223〉artificial sequence

<400>1

atacttaagc?gagaaaaaag?gttctggttc?tggtcatcat?catcatcatc?atgatgacga 60

tgaca 65

<210>2

<211>46

<212>DNA

＜213〉artificial sequence

<400>2

atacttaagc?gagaaaaaag?gttctggttc?tggtcatcat?catcat?46

<210>3

<211>54

<212>DNA

＜213〉artificial sequence

<400>3

ttgcttatgc?tttgcttgtt?tgtcatcgtc?atcatgatga?tgatgatgat?gacc 54

<210>4

<211>30

<212>DNA

＜213〉artificial sequence

<400>4

gacgatgaca?aacaagcaaa?gcataagcaa 30

<210>5

<211>29

<212>DNA

＜213〉artificial sequence

<400>5

ttcggatcct?tagcgacagc?cacaacctt 29

<210>6

<211>957

<212>DNA

＜213〉synthetic

<400>6

atggcgcagt?atgaagatgg?taaacagtac?actaccctgg?aaaaaccggt?agctggcgcg 60

ccgcaagtgc?tggagttttt?ctctttcttc?tgcccgcact?gctatcagtt?tgaagaagtt 120

ctgcatattt?ctgataatgt?gaagaaaaaa?ctgccggaag?gcgtgaagat?gactaaatac 180

cacgtcaact?tcatgggtgg?tgacctgggc?aaagatctga?ctcaggcatg?ggctgtggcg 240

atggcgctgg?gcgtggaaga?caaagtgact?gttccgctgt?ttgaaggcgt?acagaaaacc 300

cagaccattc?gttctgcttc?tgatatccgc?gatgtattta?tcaacgcagg?tattaaaggt 360

gaagagtacg?acgcgacgtg?gaacagcttc?gtggtgaaat?ctctggtcgc?tcagcaggaa 420

aaagctgcag?ctgacgtgca?attgcgtggc?gttccggcga?tgtttgttaa?cggtaaatat 480

cagctgaatc?cgcagggtat?ggataccagc?aatatgtttg?ttcagcagta?tgctgataca 540

gtgaaatact?taagcgagaa?aaaaggttct?ggttctggtc?atcatcatca?tcatcatgat 600

gacgatgaca?aacaagcaaa?gcataagcaa?cggaagaggc?tgaaatcctc?ctgcaaaaga 660

catccgctgt?atgtggattt?tagcgatgtg?ggctggaacg?attggattgt?ggcgccgccg 720

ggctatcatg?cgttttattg?ccatggcgaa?tgcccgtttc?cgctggcgga?tcatctgaac 780

tccaccaacc?atgcgattgt?gcagaccctg?gtgaactctg?tgaacagcaa?aattccgaaa 840

gcgtgctgtg?tgccgaccga?actgagcgcg?atttcgatgc?tgtatctgga?tgaaaacgaa 900

aaagtggtgc?tgaaaaacta?tcaggatatg?gtggtggaag?gttgtggctg?tcgctaa 957

<210>7

<211>318

<212>PRT

＜213〉synthetic

<400>7

Met?Ala?Gln?Tyr?Glu?Asp?Gly?Lys?Gln?Tyr?Thr?Thr?Leu?Glu?Lys?Pro

1 5 10 15

Val?Ala?Gly?Ala?Pro?Gln?Val?Leu?Glu?Phe?Phe?Ser?Phe?Phe?Cys?Pro

20 25 30

His?Cys?Tyr?Gln?Phe?Glu?Glu?Val?Leu?His?Ile?Ser?Asp?Asn?Val?Lys

35 40 45

Lys?Lys?Leu?Pro?Glu?Gly?Val?Lys?Met?Thr?Lys?Tyr?His?Val?Asn?Phe

50 55 60

Met?Gly?Gly?Asp?Leu?Gly?Lys?Asp?Leu?Thr?Gln?Ala?Trp?Ala?Val?Ala

65 70 75 80

Met?Ala?Leu?Gly?Val?Glu?Asp?Lys?Val?Thr?Val?Pro?Leu?Phe?Glu?Gly

85 90 95

Val?Gln?Lys?Thr?Gln?Thr?Ile?Arg?Ser?Ala?Ser?Asp?Ile?Arg?Asp?Val

100 105 110

Phe?Ile?Asn?Ala?Gly?Ile?Lys?Gly?Glu?Glu?Tyr?Asp?Ala?Thr?Trp?Asn

115 120 125

Ser?Phe?Val?Val?Lys?Ser?Leu?Val?Ala?Gln?Gln?Glu?Lys?Ala?Ala?Ala

130 135 140

Asp?Val?Gln?Leu?Arg?Gly?Val?Pro?Ala?Met?Phe?Val?Asn?Gly?Lys?Tyr

145 150 155 160

Gln?Leu?Asn?Pro?Gln?Gly?Met?Asp?Thr?Ser?Asn?Met?Phe?Val?Gln?Gln

165 170 175

Tyr?Ala?Asp?Thr?Val?Lys?Tyr?Leu?Ser?Glu?Lys?Lys?Gly?Ser?Gly?Ser

180 185 190

Gly?His?His?His?His?His?His?Asp?Asp?Asp?Asp?Lys?Gln?Ala?Lys?His

195 200 205

Lys?Gln?Arg?Lys?Arg?Leu?Lys?Ser?Ser?Cys?Lys?Arg?His?Pro?Leu?Tyr

210 215 220

Val?Asp?Phe?Ser?Asp?Val?Gly?Trp?Asn?Asp?Trp?Ile?Val?Ala?Pro?Pro

225 230 235 240

Gly?Tyr?His?Ala?Phe?Tyr?Cys?His?Gly?Glu?Cys?Pro?Phe?Pro?Leu?Ala

245 250 255

Asp?His?Leu?Asn?Ser?Thr?Asn?His?Ala?Ile?Val?Gln?Thr?Leu?Val?Asn

260 265 270

Ser?Val?Asn?Ser?Lys?Ile?Pro?Lys?Ala?Cys?Cys?Val?Pro?Thr?Glu?Leu

275 280 285

Ser?Ala?Ile?Ser?Met?Leu?Tyr?Leu?Asp?Glu?Asn?Glu?Lys?Val?Val?Leu

290 295 300

Lys?Asn?Tyr?Gln?Asp?Met?Val?Val?Glu?Gly?Cys?Gly?Cys?Arg

305 310 315

<210>8

<211>15

<212>PRT

<213>Unknown

<220>

＜223〉synthetic

<400>8

Gln?Ala?Lys?His?Lys?Gln?Arg?Lys?Arg?Leu?Lys?Ser?Ser?Cys?Lys

1 5 10 15

Claims

1. the preparation method of bone morphogenic protein BMP-2-2 mature peptide is characterized in that described method comprises following sequential steps:

(1) the 2nd～18 amino acid encoding gene of deletion DsbA gene signal peptide, the codon that keeps first methionine(Met) is as initiator codon, obtain the encoding dna fragmentation of the DsbA that do not contain signal peptide is reconstituted in this fragment in the expression vector, obtains recombinant plasmid 1;

(6) the proteolytic enzyme enzyme is cut the BMP-2 fusion rotein, obtains described bone morphogenic protein BMP-2-2 mature peptide through separation.

2. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 1 is characterized in that the DsbA gene is from plasmid pET39 described in the described step (1).

3. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 1 is characterized in that host strain is e. coli bl21 (DE3) in the described step (3).

4. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 1 is characterized in that being expressed under the shortage oxidation envrionment conditions of goal gene carried out in the described step (4).

5. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 1 is characterized in that in the described step (6) that proteolytic enzyme is one of following: enteropeptidase, recombinant enterokinase light chain.

6. as the preparation method of one of claim 1～5 described bone morphogenic protein BMP-2-2 mature peptide, it is characterized in that in the described step (2), between the DNA of the complete mature peptide of bone morphogenic protein BMP-2 in the recombinant plasmid 2-2 and the downstream of DsbA gene, also reorganization has the sequence of coding " Linker-6His-proteolytic enzyme recognition site ".

7. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 6 is characterized in that described proteolytic enzyme recognition site is enteropeptidase or thrombin.

8. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 1, it is characterized in that described method in the following order step carry out:

9. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 8, it is characterized in that described method in the following order step carry out:

(5) after the fermentation ends, centrifugal collection engineering bacteria cell, centrifugal behind the broken bacterium, the collection supernatant liquor is crossed and is contained Ni ²⁺Chelate column, with the pre-wash-out of 50mM imidazole buffer, containing the buffer solution elution target protein of imidazoles 120～150mM, collect elutriant;

(6) in the elutriant that contains the BMP-2 fusion rotein, add the diethylamine tetraacethyl of final concentration 0.1～1mM, and enteropeptidase 5U/mL, carry out enzyme under 25 ℃ to cut, the proteolytic enzyme enzyme is cut fusion rotein, obtains described bone morphogenic protein BMP-2-2 mature peptide through separation.

10. the preparation method of bone morphogenic protein BMP-2-2 mature peptide as claimed in claim 8, it is characterized in that described method in the following order step carry out:

(1) be masterplate with the plasmid pET39 that contains the DsbA gene,

Primer: DsbA-1:5 '-AAGCATATGGCGCAGTATGAAGAT-3 ',

DsbA-2：5’-TCGCTTAAGTATTTCACTGT-3’，

PCR parameter: 94 ℃ of 3min

3 circulations of 72 ℃ of 1min of 40 ℃ of 30s of 94 ℃ of 30s

27 circulations of 72 ℃ of 1min of 48 ℃ of 30s of 94 ℃ of 30s

72℃ 10min

4℃ 10min

(2) the DNA fragment of " Linker-6His-Eksite ": the ATA that will encode CTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCATCATCATCATCATCATGATGACGATGACA AA, wherein CTTAAGBe the BspTI recognition sequence,, obtain the dna fragmentation of coding " Linker-6His-Eksite-BMP-2 " with the DNA splicing of coding bone morphogenic protein BMP-2-2 mature peptide:

Primer Linker-1:5 '-ATA CTTAAGCGAGAAAAAAGGTTCTGGTTCT

GGTCATCATCATCAT-3’

Primer Linker-2:5 '-TTGCTTATGCTTTGCTTGTTTGTCATCGTCATC

ATGATGATGATGATGATGACC?3’

Primer BMP2-1:5 '-GACGATGACAAA CAAGCAAAGCATAAGCAA-3 '

Primer BMP2-2:5 '-TTCGGATCCTTAGCGACAGCCACAACCTT-3 '

Getting concentration is primer Linker-1 and each 2uL of Link-2 of 10uM, adds 2uL dNTP, and 5uL 10 * DNA Polymerase Buffer mends ddH ₂O to 50uL reaction system, boiling water bath 10min puts into 50 ℃ of water-bath cooling 3min, adds 2.5U Pfu DNA polymerase, changes 72 water-bath 10min over to, makes two primers template each other, forms the dna double chain, obtains reaction product A;

PCR parameter: 94 ℃ of 3min

30 circulations of 72 ℃ of 1min of 60 ℃ of 30s of 94 ℃ of 30s

72℃ 10min

4℃ 10min；

(6) after the fermentation ends, centrifugal collection engineering bacteria cell, centrifugal after the carrying out ultrasonic bacteria breaking, the collection supernatant liquor is crossed and is contained Ni ²⁺Chelate column, with the pre-wash-out of 50mM imidazole buffer,, collect elutriant with Tris-HCl, the pH7.8 buffer solution elution that contains the 150mM imidazoles;

(7) in the elutriant that contains the BMP-2 fusion rotein, add the diethylamine tetraacethyl of final concentration 1mM, and enteropeptidase 5U/mL, carry out enzyme under 25 ℃ to cut, the proteolytic enzyme enzyme is cut fusion rotein, obtains described bone morphogenic protein BMP-2-2 mature peptide through separation.