Research On Expression,Purification And Activity Of Recombinant Human Bone Morphogenetic Protein 7-2

Bone morphogenetic protein(BMP)is a factor which can induce osteoclast formation.BMPs possess a wide prospect of clinical application because it play a key role in development,regeneration and repairing of bone.Now,BMPs that applied in clinic are obtained from animal cells,however,the low yield and high cost of BMP prepared by this method result the treatment cost is very high.In this study,the expression vector pBMP7-2 was constructed and then rhBMP7-2 fusion protein was expressed in E.coli.After a simplified dialysis,the refolding rhBMP7-2 were achieved.A variety of activity tests had validated their functional efficacy both in vivo and in vitro.The main research contents and results are as follows:(1)The BMP7 and BMP2 mature peptide genes were linked by a flexible peptide linker(GlyGlyGlyGlySer)3 gene by the fusion PCR.The DNA fragments were cloned into expression vector pHis-NusA and pBV220,and named as pBMP2-7,pBMP7-2,pBV220-BMP2-7 and pBV220-BMP7-2,respectively.Expressed in E.coli,the protein rhBMP7-2,rhBMP2-7 were all existed in the form of inclusion body.(2)After renatured by dialysis,only part of rhBMP7-2 can be renatured successfully,so we choose it for the further study.After purified,the inclusion body were renatured by dialysis method.A series of experiments were performed to test the refolding conditions,then the system and steps were adopted:the target protein was denatured by denaturing solution with 8M urea,and dissolved at the concentration of 0.2 mg/ml,then the bag with the protein was introduced to the dialysate buffer with 2 M urea,and the gradient 8M-2M was dialyzed overnight at 4?,then refolded with a urea gradient of 1 M-0 M-0 M for 3 h for every gradient,the dialysate buffer included 100 mM Na3PO4,pH 7.4,250 mM NaCl,10%glycerol,4 mM cysteine,0.5 mM cystine.At last,the concentration of rhBMP7-2 fusion protein in supernatant was 96 ?g/ml determined by Bradford method.(3)MC3T3-E1 and C2C12 cells were induced by different concentrations of rhBMP7-2 heterodimer fusion protein.The alkaline phosphatase(ALP)activity had been adopted as a early marker of bone-formation,The ALP of two kinds cells was maximum at the protein concentration of 0.2 ?g/mL,and the working concentration of the rhBMP7-2 dimer fusion protein was one fifth of the rhBMP2 homodimer when the ALP rearched maximum.Alizarin red staining were proformed to detect calcium deposits of MC3T3-E1 and C2C12 cells induced by rhBMP7-2,and the calcium deposits was a later maker of bone-formation.The result indicated that the rhBMP7-2 fusion protein can induced the cells to produced more red spots than rhBMP2 homodimer.Rat osteogenesis models were used to test the activity of rhBMP7-2.This experiment indicated the rhBMP7-2 fusion protein obtained from the refolding process contains ectopic osteogenic activity.