CN102336829B - Method for producing recombinant human bone morphogenetic protein-2 mature peptide - Google Patents
Method for producing recombinant human bone morphogenetic protein-2 mature peptide Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for producing recombinant human bone morphogenetic protein-2 mature peptide. The method comprises the following steps: sampling a recombinant human bone morphogenetic protein-2 mature peptide solution with good renaturation into a balanced hydrophobic chromatography column, then performing a stepped-gradient elution which gradually reduces the salinity by an elution buffer solution, and collecting target peaks. In order to further enhance the protein purity of the target peaks, the purification method can be a multi-step hydrophobic interaction chromatography or be used by combining with the ion exchange chromatography. The method has the advantages of simple operation, lower cost, higher purification yield (more than 20%), higher purity (SDS-PAGE, HPLC and HPCE are greater than 97%) and the like, and is suitable for producing the recombinant human bone morphogenetic protein family.
Description
Technical field
The present invention relates to protein purification field, specifically, relate to the production method for purifying of a kind of recombinant human bone morphogenesis protein-2 (rhBMP-2) mature peptide.
Background technology
Nineteen sixty-five, American physician Urist (Urist MR.Bone:formation byautoinduction.Science, 1965,150 (698): 893-899) the bone interstitial of discovery decalcification has ectopic, and by newfound have induction new osteoplastic protein called after Delicious peptide (Bone Morphogenetic protein, BMP).Subsequently, found again numerous BMP, except BMP-1, they are all TGF-beta superfamily members, no matter on gene or protein structure, or all closely similar on biological function.Wherein the Study on Osteogenesis of BMP-2 is maximum, and study on the industrialization is also extensive.
People BMP-2 total length is 396 amino-acid residues, and what have osteogenesis is mature peptide, is made up of 114 amino acid, on the asparagine residue of 55, has glycosylation site, but whether glycosylation does not affect its biological activity.In addition BMP-2 hydrophobicity is very strong, is dissolved in hardly all kinds of SOLVENTS, and its activity form exists with dimeric forms, and wherein 3 pairs of disulfide linkage of the each formation of monomer form a pair of interchain disulfide bond between two monomers.Whether the correct pairing of disulfide linkage directly affects its biological activity and stability.Internal and external test proves that BMP-2 has the ability (Riley, the EH that promote osteoblast differentiation and the outer skeletonization of inductor; Lane, JM; Urist, MR, et al.Bone morphogenetic protein-2:Biology and applications.ClinOrthop Relat Res.1996Mar; (324): 39-46).But the content of natural BMP-2 in various tissues is extremely low, need to adopt the method for gene recombination to produce in a large number.
Aspect the study on the industrialization of BMP-2, be mainly that carrier for expression of eukaryon and intestinal bacteria are two kinds of gene recombination schemes of prokaryotic expression system of representative.The domestic also unrealized industrialization of carrier for expression of eukaryon, 1988, (the Wozney J.M. such as wozney, Rosen V., Celeste A.J.etal.Novel regulators of bone formation:molecular clones and activities.Science 16 December 1988, 242 (4885): 1528-1534.) obtained the BMP-2 of COS-1 eukaryotic expression, 1992, Israel DI has obtained BMP-2 (the Israel DI of CHO eukaryotic expression, Nove J, Kerns KM, Moutsatsos IK, Kaufman RJ.Expressionand characterization of bone morphogenetic protein-2 in Chinese hamsterovary cells.Growth Factors 1992, 7:139-50), the INFUSE bone migration agent of Medtronic SofamorDanek company of U.S. development and LT-CAGE Lumbar Fusion apparatus obtain FDA approval in July, 2002 and are used for the treatment of intervertebral disk retrogression disease (McKay WF, Peckham SM, Badura JM.A comprehensive clinical review ofrecombinant human bone morphogenetic protein-2 (INFUSE Bone Graft) .Int Orthop.2007 Dec, 31 (6): 729-34), the InductOs (TM) of Wyeth company exploitation (rhBMP-2/ACS) obtains the approval of European Commission (EC) in September, 2002, for treatment (the Govender S of the acute fracture of tibia of being grown up, Csimma C, GenantHK, et al.Recombinant human bone morphogenetic protein-2 fortreatment of open tibial fractures:a prospective, controlled, randomizedstudy of four hundred and fifty patients.J Bone Joint Surg Am.2006Jun, 88 (6): 1258-65).The expression system of INFUSE bone migration agent and InductOs is all Chinese hamster ovary celI, and expression amount is low, and production cost is very high, and is not suitable for Chinese development present situation.
Aspect prokaryotic expression system, Zhao Ming (Zhao Ming in 1994, Wang Huixin, Zhou Tingchong. expression and the induced osteogenesis activity [J] thereof of recombinant human bone morphogenesis protein-2 mature peptide in intestinal bacteria. Chinese biological chemistry and molecular biosciences journal, 1994, 10 (03) 319-324), (the Lu Zifan such as nineteen ninety-five Chen Sumin, Liu Xinping, Chen Sumin, Chen Nanchun. high expression level and the determination of activity of Human Bone Morphogenetic Proteins-4's 2 carbon teminal peptides in intestinal bacteria. Journal of the Fourth Military Medical University, 1995, 16 (6): 429-431) completed the high efficient expression in intestinal bacteria of BMP-2, expression product is inclusion body, therefore need to carry out external renaturation.
(the Gross G such as German Vallejo in 2002, Weich HA, Rinas is and purification of bone morphogenetic protein-2 producedas inclusion bodies in high-cell-density cultures of recombinantEscherichia coli.J Biotechnol.2002 Mar 28 U.2002.Renaturation; 94 (2): 185-94) realized scale operation intestinal bacteria, every liter of nutrient solution can obtain 75 grams of dry cell weights, the purifying BMP-2 of 750mg.Dilution method is carried out renaturation and after 4 days, is carried out heparin column affinitive layer purification.But for BMP-2, heparin column carrying capacity is too little, average every milliliter of heparin affinity gel is less than the binding capacity of 2mg.The purge process exchange buffering liquid of need to dialysing, also needs ultrafiltration and concentration after purifying.
2004, (Vallejo LF, the U.2004.Optimizedprocedure for renaturation of recombinant human bone morphogeneticprotein-2 at high protein concentration.Biotechnol Bioeng.2004 Mar20 of Rinas such as Germany Vallejo; 85 (6): 601-9) successfully developed the renaturation scheme of high density, can obtain the activated protein up to 0.87mg/mL, renaturation yield is up to 43%, but the reagent 2-cyclohexyl ethyl sulfonic acid (CHES) using, Nicotinicum Acidums (NA) etc. are all very expensive, and purification schemes is still heparin column affinity chromatography.
Domestic grandson investigator courageously waits and has carried out renaturation technical study (Sun Fenyong in 2004, Wang Ju, Sun Jinhua, Dai Yun, Qiu Chuiyuan, Hong An, expression, purifying and the renaturation (English) of recombinant human bone morphorgenetic protein-2 in intestinal bacteria, Chinese Journal of Pathophysiology, 2005,21 (8): 1480-1485); The few China of Yao in 2005 waits and utilizes the first purifying inclusion body of hydroxyapatite column chromatography metaprotein to carry out renaturation (Yao, SH again; Zhang, LL; Cheng, SY, etal..2005.Refolding and Purification of rhBMP-2 Expressed as InclusionBodies in E.coli with Hydroxyapatite Chromatography.KEYENGINEERING MATERIALS.2005, VOL 288-289:661-664); Within 2008, the Chen Su people have carried out the renaturation (Wang Fuli under High Concentration Situation, Chen Sumin, Chen Nanchun, the Zhao Wei renaturation of .rhBMP-2m under High Concentration Situation of admiring. Journal of the Fourth Military Medical University, 2008,29 (12): 1071-1074), although protein concentration is up to 6mg/mL, but BMP-2 metaprotein need to carry out sex change again after ion exchange chromatography purifying, and dialysis method renaturation scale is less, is unfavorable for suitability for industrialized production.
Investigator adopts heparin column affinity chromatography to carry out the purifying of BMP-2 activated protein (Ruppert, R mostly both at home and abroad; Hoffmann, E; Sebald, W.1996.Human bonemorphogenetic protein 2 contains a heparin-binding site which modifiesits biological activity.Eur J Biochem., 1996 Apr 1; 237 (1): 295-302), do not have the commercial scale production of real meaning.
2006, Xu is put (CN 200510050610.3) and has developed the purifying process of ion-exchange and hydrophobic interaction chromatography method, purification schemes is mainly: ion-exchange is Q Sepharose FF, use the 2-cyclohexyl ethyl sulfonic acid (CHES) of 25-100mM, pH7.5-9.5,1-3M urea, the damping fluid of 5-20% dimethyl formamide composition is cooked balance liquid, the gradient elution that the sodium chloride concentration of 0.1-1.0M increases progressively.Adopt hydrophobic interaction chromatography to adopt phenyl sepharose gel, with the 2-cyclohexyl ethyl sulfonic acid of 25-100mM, pH7.5-9.5,1-3M urea, 5-20% dimethyl formamide, the damping fluid of 1-4M sodium-chlor composition is cooked balance liquid, carries out the gradient elution that salt concn is successively decreased.This scheme is still used the CHES of higher concentration, and urea concentration is also higher, and dimethyl formamide maximum concentration is 20%, and the rate of recovery is greater than 15% generally, and purity is greater than 95%, but production cost is higher.
2007, Liu Guoan etc. (CN200610049151.1) have developed two step ion exchange chromatography purifying process (Q sepharose and CM sepharose), purification schemes is: first renaturation solution carries out Q post anion-exchange chromatography above with 10 times of low salt buffer dilutions, with 20mM trishydroxymethyl ammonia methane-HCl, pH8.5 damping fluid is cooked balance liquid, carry out the gradient elution (target protein is in 0.18M gradient) that sodium chloride concentration progressively increases progressively, then dilute 5 times and carry out CM post cation-exchange chromatography, adopt 15mM phosphoric acid, pH6.5 balance liquid is cooked balance liquid, carry out the gradient elution (target protein is in 0.21M gradient) that sodium chloride concentration progressively increases progressively.But because renaturation solution concentration is only 0.08-0.1mg/mL; when purifying, first need a large amount of dilution (10 times); loading volume is very large; be unfavorable for large-scale production; moreover the ion exchange chromatography of two steps all do not add solubility promoter, when wash-out the stripping of BMP-2 target protein very slow, even do not dissolve; cause purification effect variation, suitability for industrialized production that can not be real.
Summary of the invention
Also cannot realize the technological deficiency of efficient suitability for industrialized production in order to solve prior art Restruction human bone morphogenesis protein-2 (rhBMP-2) mature peptide, the invention provides a kind of be different from aforesaid method, easy and simple to handle, with low cost, be applicable to extensive production high purity of amplifying, the separation purification method of highly active recombinant human bone morphogenesis protein-2 mature peptide.
Protokaryon system construction and the expression of recombinant human bone morphogenesis protein-2 (rhBMP-2) mature peptide, the existing document of preparation, solubilising and refolding method of inclusion body is fully open, can put (CN01116754.8 with reference to Xu in detail; CN 200510050610.3) and Liu Guoan etc. (CN200610049151.1), above-mentioned document is all incorporated herein by reference at this.Recombinant human bone morphogenesis protein-2 (rhBMP-2) mature peptide can be 115 amino-acid residues that 114 amino-acid residues of total length or N-terminal add methionine(Met); Also can be the mature peptide of truncation type, a truncation type mature peptide (bibliographic reference CN01116754.8) that example is 108 amino-acid residues, concrete sequence is: MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAI VQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR
Inclusion body is after renaturation, and above-mentioned recombinant human bone morphogenesis protein-2 (rhBMP-2) mature peptide monomer exists with dimeric forms, and wherein 3 pairs of disulfide linkage of the each formation of monomer form a pair of interchain disulfide bond between two monomers.
The separation purification method that the invention provides a kind of recombinant human bone morphogenesis protein-2 (rhBMP-2) mature peptide, the method comprises the following steps:
(1) recombinant human bone morphogenesis protein-2 mature peptide solution good renaturation is splined on to the hydrophobic chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, 1-3M sodium-chlor or ammonium sulfate, pH is 7.0-9.0;
(2) carry out with elution buffer the stagewise gradient wash-out that salt concn progressively reduces, collect target peak, described elution buffer comprises 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, and pH is 7.0-9.0.
In aforesaid method, hydrophobic chromatography post medium can (include but not limited to Phenyl Sepharose 6 Fast Flow (low sub) from phenyl sepharose gel, Phenyl Sepharose 6Fast Flow (high sub), Phenyl Sepharose High Performance), butyl-agarose gel (includes but not limited to Butyl Sepharose 4Fast Flow, Butyl-SSepharose 6 Fast Flow, Butyl Sepharose High Performance), octyl sepharose gel (including but not limited to Octyl Sepharose 4 Fast Flow), ethyl polymer resin (SOURCE ETH), sec.-propyl polymer resin (SOURCE 15ISO), phenyl polymer resin (SOURCE 15PHE).
In aforesaid method, level pad and elution buffer can further contain 10-50mM trishydroxymethyl ammonia methane-HCl, better to maintain buffer solution system pH as 7.0-9.0.
For further improving the purity of protein of target peak, purification process can be multistep hydrophobic interaction chromatography or combine use with ion exchange chromatography.
As one of preferred version, purification process can be multistep hydrophobic interaction chromatography.The target peak that one step hydrophobic chromatography obtains is again through 1-2 hydrophobic chromatography, more than can effectively improving purity of protein to 97%.
As one of preferred version, purification process can be hydrophobic chromatography and anion-exchange chromatography coupling, chromatography media is Q sepharose Rapid Flow (Q-sepharose Fast Flow), Q sepharose high resolving power (Q-sepharose HP) or Q polymer resin (SOURCE 15Q, SOURCE 30Q), level pad comprises 10-50mM trishydroxymethyl ammonia methane-HCl, 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea and 2-10%N, dinethylformamide, pH is 8.0-9.0; Elutriant is the level pad that has added 1-3M NaCl.The method, except above-mentioned hydrophobic chromatography step, further comprises following steps:
(1) target peak hydrophobic chromatography being obtained is after level pad dilution, be splined on the anion-exchange chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-50mM trishydroxymethyl ammonia methane-HCl, 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea and 2-10%N, dinethylformamide, pH is 8.0-9.0;
(2) carry out wash-out by the gradient that elution buffer carries out salt concn and progressively increases, collect main peak, described elution buffer comprises 10-50mM trishydroxymethyl ammonia methane-HCl, 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea and 2-10%N, dinethylformamide, 1-3M NaCl, pH is 8.0-9.0.
As one of preferred version, purification process can be hydrophobic chromatography and cation-exchange chromatography coupling, chromatography media is carboxymethyl sepharose Rapid Flow (CM-Sepharose Fast Flow), level pad comprises 10-50mM SODIUM PHOSPHATE, MONOBASIC-HCl, 10-30mM 2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, pH is 6.0-6.5.Elutriant is the level pad that has added 1-3M NaCl.The method, except above-mentioned hydrophobic chromatography step, further comprises following steps:
(1) target peak hydrophobic chromatography being obtained is after level pad dilution, be splined on the cation-exchange chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-50mM SODIUM PHOSPHATE, MONOBASIC-HCl, 10-30mM2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, pH is 6.0-6.5;
(2) carry out wash-out by the gradient that elution buffer carries out salt concn and progressively increases, collect main peak, described elution buffer comprises 10-50mM SODIUM PHOSPHATE, MONOBASIC-HCl, 10-30mM2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, 1-3MNaCl, pH is 6.0-6.5.
The present invention, owing to having adopted above-mentioned scheme, has successfully realized the large-scale industrialization of recombinant human bone morphogenesis protein-2 mature peptide and has produced.The present invention gropes by test of many times, find to adopt urea and the DMF of high density to make solubility promoter, to increase the polarity of recombinant human bone morphogenesis protein-2 mature peptide, in hydrophobic chromatography process, will reduce and the conjugation of hydrophobic medium, thereby reduce efficiency and the resolution of protein purification.The present invention is by systematic study, the unexpected composition of finding by adjusting purifying damping fluid, uses the urea of low concentration and DMF to make solubility promoter, and has reduced the consumption of CHES, efficiency and the resolution that can effectively improve protein purification, the purity of target protein is also higher.And owing to having reduced the consumption of urea, DMF and CHES, production cost can reduce more than 50%.Compared with the invention (CN200510050610.3) that this purification process is put with Xu, there is the advantages such as operation is easier, cost is cheaper, purification yield higher (being greater than 20%), purity higher (SDS-PAGE, HPLC and HPCE are all greater than 97%).
This method is applicable to the industrialized purification of recombinant human bone morphogenetic protein family and produces.
Brief description of the drawings
Fig. 1 hydrophobic interaction chromatography purifying BMP-2 tomographic map.Wherein 1 is UV280nm, and 2 is UV260nm, and 3 is solution conductivity, and 4 is gradient.Peak 1 is gradient 1, and peak 2 is gradient 2, and peak 1 and peak 2 contain more target protein, and peak 3 is gradient 3, contains less target protein, and peak 4, for cleaning peak, is mainly foreign protein and monomeric protein, polymer etc.
The typical rhBMP-2 electrophorogram of Fig. 2 (silver dyes).Diagram Lane1: renaturation solution, lane2: peak 1 and peak 2 merge, lane3: peak 1, lane4: peak 2, lane5: peak 3, lane6: peak 4, lane7: protein molecular weight marker (from being respectively up and down 97.4,, 66.1,43,, 31,20,14.4KDa).
The typical rhBMP-2 electrophorogram of Fig. 3 (silver dyes).Diagram Lane1: renaturation solution, lane2: a step drainage column elution peak 1, lane3: a step drainage column wash-out peak-to-peak 2, lane4: the hydrophobic wash-out main peak of two steps, lane5 a: step is hydrophobic, main peak after a step anionresin, lane6 a: step is hydrophobic, main peak after a step cationic exchange, lane7: protein molecular weight marker (from being respectively up and down 97.4,66.1,43,31,20,14.4KDa), 8: two hydrophobic elution peak concentrating samples of step of lane.
The capillary electrophoresis purity check of Fig. 4 rhBMP-2, UV214nm detects.Adopting area normalization method calculated purity is 97.4%.
The capillary electrophoresis purity check of Fig. 5 rhBMP-2, UV214nm detects.Adopting area normalization method calculated purity is 98.0%.
The capillary electrophoresis purity check of Fig. 6 rhBMP-2, UV214nm detects.Adopting area normalization method calculated purity is 98.5%.
Embodiment
Below in conjunction with embodiment, the present invention is further described, what in embodiment, relate to is the BMP-2 mature peptide of 108 amino-acid residues of truncation type.Those skilled in the art will appreciate that because it is similar to the BMP-2 mature peptide character of total length, thereby be also suitable for the separation and purification of the BMP-2 mature peptide of full-length.
The solubilising of example 1 inclusion body
With 1 gram of inclusion body: the ratio of 20mL lysate by solubilization of inclusion bodies in lysate (7M Guanidinium hydrochloride, 50mM Tris-HCl, pH8.0,1mM EDTA, 10mM DTT) at room temperature stir above or 4 DEG C of stirrings in 2 hours and spend the night.Then centrifugal 20min at 12000rpm, 4 DEG C, abandons precipitation and gets centrifuged supernatant.Adopt Bradford method to measure protein concentration.
Or use following methods solubilising.With 1 gram of inclusion body: the ratio of 20mL lysate by solubilization of inclusion bodies in lysate (8M urea, 50mM Tris-HCl, pH8.5,1mM EDTA, 10mM DTT) at room temperature stir above or 4 DEG C of stirrings in 2 hours and spend the night.Then centrifugal 20min at 12000rpm, 4 DEG C, abandons precipitation and gets centrifuged supernatant.Adopt Bradford method to measure protein concentration.
The renaturation of example 2 inclusion bodys
Adopt dilution method renaturation.Renaturation solution consists of: 100mM Tris-HCl, 0.5M L-Arg, 0.5M Urea, 1M NaCl, 2mM EDTA, pH8.5-9.5,1mM GSSG, 5mM reduced glutathion.Inclusion body solubilising liquid is slowly joined in renaturation solution, and control final concentration of protein is 0.05-0.3mg/mL, renaturation 1-20 days under 4-20 degree isoperibol.
The hydrophobic interaction chromatography of example 3 renaturation solutions
First adopt the hydrophobic interaction chromatography that salt concn is low, chromatographic stuffing is phenylsepharose Fast Flow, and balance liquid is phe-A:0.5M urea, 25mM CHES, 15mMTris, pH8.5,5%N, dinethylformamide, 1M NaCl.Elutriant is phe-B:0.5Murea, 25mM CHES, 15mM Tris, pH8.5,5%N, dinethylformamide.First use balance liquid phe-A balance, the direct loading of renaturation solution, continues to be flushed to ultraviolet with level pad phe-A and arrives baseline after loading, then carry out with elution buffer phe-B the gradient that salt concn progressively reduces and carry out wash-out, collection main peak.
For further improving purity, can continue to adopt the high hydrophobic interaction chromatography of salt concn to collecting main peak.Chromatographic stuffing is phenyl sepharose Fast Flow, and balance liquid is phe-3A:0.5Murea, 25mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide, 3MNaCl.Elutriant is phe-3B:0.5M urea, 25mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide.First use balance liquid phe-3A balance, the hydrophobic interaction chromatography elution samples that above-mentioned salt concn is low is added NaCl to loading after 3M, after loading, continue to be flushed to ultraviolet with level pad phe-3A and arrive baseline, then carry out with elution buffer phe-3B the stagewise gradient that salt concn progressively reduces and carry out wash-out (35%, 65%, 85%B), substep is collected corresponding main peak.SDS-PAGE Gel Electrophoresis Silver dyes analytic sample purity.As shown in Figure 1, electrophorogram as depicted in figs. 1 and 2 for typical tomographic map.Feed intake and count from renaturation solution, renaturation yield is greater than 50%, and the yield of ultimate aim albumen is 21%, uses capillary electrophoresis analysis purity, and purity is 97.4% (Fig. 4).
Example 4 hydrophobic interaction chromatographies and anion-exchange chromatography coupling purifying
First adopt hydrophobic interaction chromatography, chromatographic stuffing is phenyl sepharose Fast Flow, and balance liquid is phe-3A:0.8M urea, 15mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide, 3M NaCl.Elutriant is phe-3B:0.8M urea, 15mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide.First use balance liquid phe-3A balance, the direct loading of renaturation solution, after loading, continue to be flushed to ultraviolet with level pad phe-3A and arrive baseline, then carry out with elution buffer phe-3B the stagewise gradient that salt concn progressively reduces and carry out wash-out (35%, 65%, 85%B), substep is collected corresponding main peak.
Then carry out anion-exchange chromatography, Q-Sepharose Fast Flow, balance liquid is Q-A:0.8M urea, 15mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide.Elutriant is Q-B:0.8M urea, 15mM CHES, 15mM Tris, pH8.5,4%N, dinethylformamide, 1MNaCl.First use the good pillar of balance liquid Q-A balance, rhBMP-2 sample makes specific conductivity lower than 6ms/cm with 10 times of balance liquid Q-A dilutions above, then loading, after loading, continue to be flushed to ultraviolet with level pad Q-A and arrive baseline, then carry out wash-out by the gradient that elution buffer Q-B carries out salt concn and progressively increases, collect main peak.SDS-PAGE Gel Electrophoresis Silver dyes analysis (Fig. 3) and capillary electrophoresis analysis sample purity is 98.0% (Fig. 5).
Example 5 hydrophobic interaction chromatographies and cation-exchange chromatography coupling purifying
First adopt hydrophobic interaction chromatography, chromatographic stuffing is phenyl sepharose Fast Flow, and balance liquid is phe-3A:0.5M urea, 25mM CHES, 15mM Tris, pH8.5,2%N, dinethylformamide, 3M NaCl.Elutriant is phe-3B:0.5M urea, 25mM CHES, 15mM Tris, pH8.5,2%N, dinethylformamide.First use balance liquid phe-3A balance, the direct loading of renaturation solution, after loading, continue to be flushed to ultraviolet with level pad phe-3A and arrive baseline, then carry out with elution buffer phe-3B the stagewise gradient that salt concn progressively reduces and carry out wash-out (35%, 65%, 85%B), substep is collected corresponding main peak.
Then carry out cation-exchange chromatography purifying, chromatographic stuffing is CM-Sepharose FastFlow, and balance liquid is CM-A:0.5M urea, 25mM CHES, 15mM NaAc-HAc, 2%N, dinethylformamide, pH6.5.Elutriant is CM-B:0.5M urea, 25mMCHES, 15mM NaAc-MAc, 2%N, dinethylformamide, 1M NaCl, pH6.5.First use the good pillar of balance liquid CM-A balance, hydrophobic chromatography elution samples is that specific conductivity is lower than 6ms/cm with 5 times of balance liquid CM-A dilutions above, then loading, after loading, continue to be flushed to ultraviolet with level pad CM-A and arrive baseline, then carry out wash-out by the gradient that elution buffer CM-B carries out salt concn and progressively increases, collect main peak.SDS-PAGE Gel Electrophoresis Silver dyes analysis (Fig. 3) and capillary electrophoresis analysis sample purity is 98.5% (Fig. 6).
Claims (9)
1. a production method for purifying for recombinant human bone morphogenesis protein-2 mature peptide, is characterized in that, comprises the following steps:
(1) recombinant human bone morphogenesis protein-2 mature peptide solution good renaturation is splined on to the hydrophobic chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-15mM2-cyclohexylamino ethyl sulfonic acid, 0.2-0.8M urea, 2-4%N, dinethylformamide, 1-3M sodium-chlor or ammonium sulfate and 10-50mM trishydroxymethyl ammonia methane-HCl, pH is 7.0-9.0;
(2) carry out with elution buffer the stagewise gradient wash-out that salt concn progressively reduces, collect target peak, described elution buffer comprises 10-15mM2-cyclohexylamino ethyl sulfonic acid, 0.2-0.8M urea, 2-4%N, dinethylformamide and 10-50mM trishydroxymethyl ammonia methane-HCl, pH is 7.0-9.0.
2. production method for purifying according to claim 1, is characterized in that, described hydrophobic chromatography post medium is selected from phenyl sepharose gel, butyl-agarose gel, octyl sepharose gel, ethyl polymer resin, sec.-propyl polymer resin or phenyl polymer resin.
3. production method for purifying according to claim 1, is characterized in that, described recombinant human bone morphogenesis protein-2 mature peptide is 115 amino-acid residues that 114 amino-acid residues of total length or N-terminal add methionine(Met); Or there is the truncation type mature peptide of 108 amino-acid residues, its sequence is:
MKKLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。
4. production method for purifying according to claim 1, is characterized in that, described purification process is multistep hydrophobic interaction chromatography, and the target peak that a step hydrophobic chromatography obtains, again through 1-2 hydrophobic chromatography, is collected target peak.
5. production method for purifying according to claim 1, is characterized in that, described purification process is that hydrophobic interaction chromatography and ion exchange chromatography are combined use.
6. production method for purifying according to claim 5, is characterized in that, described ion exchange chromatography is anion-exchange chromatography coupling, and chromatography media is Q-sepharose Fast Flow, Q-sepharose HP, SOURCE15Q or SOURCE30Q.
7. production method for purifying according to claim 6, is characterized in that, described method, except hydrophobic chromatography step, also comprises following steps:
(1) target peak hydrophobic chromatography being obtained is after level pad dilution, be splined on the anion-exchange chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-50mM trishydroxymethyl ammonia methane-HCl, 10-15mM2-cyclohexylamino ethyl sulfonic acid, 0.2-0.8M urea and 2-4%N, dinethylformamide, pH is 8.0-9.0;
(2) carry out wash-out by the gradient that elution buffer carries out salt concn and progressively increases, collect main peak, described elution buffer comprises 10-50mM trishydroxymethyl ammonia methane-HCl, 10-15mM2-cyclohexylamino ethyl sulfonic acid, 0.2-0.8M urea and 2-4%N, dinethylformamide, 1-3MNaCl, pH is 8.0-9.0.
8. production method for purifying according to claim 5, is characterized in that, described ion exchange chromatography is cation-exchange chromatography coupling, and chromatography media is CM-Sepharose Fast Flow.
9. production method for purifying according to claim 8, is characterized in that, described method, except hydrophobic chromatography step, also comprises following steps:
(1) target peak hydrophobic chromatography being obtained is after level pad dilution, be splined on the cation-exchange chromatography post good through balance, after loading, continue to rinse and arrive baseline with level pad, described level pad comprises 10-50mM SODIUM PHOSPHATE, MONOBASIC-HCl, 10-30mM2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, pH is 6.0-6.5;
(2) carry out wash-out by the gradient that elution buffer carries out salt concn and progressively increases, collect main peak, described elution buffer comprises 10-50mM SODIUM PHOSPHATE, MONOBASIC-HCl, 10-30mM2-cyclohexylamino ethyl sulfonic acid, 0.2-1M urea, 2-10%N, dinethylformamide, 1-3M NaCl, pH is 6.0-6.5.
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| CN1757723A (en) * | 2005-07-07 | 2006-04-12 | 徐放 | Method of producing recombination human bone morphopoiesis protein |
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