Expression Of Human Bone Morphogenetic Protein 2 In Pichia Pastoris And Determination Of Its Biological Activity

Bone morphogenetic protein 2(BMP2)which belongs to the transforming growth factor-beta(TGF-?)superfamily,one of the most potent osteogenic signaling molecules,has been demonstrated to be a strong osteoinductive factor in stimulating osteogenic and endochondral regeneration,and it has previously been used for the treatment of many bone fractures,such as bone defects,non-union fractures,spinal fusion,osteoporosis,and root canal surgery.However,isolating BMPs directly from bones usually have a series of problems,such as high cost and low production yield(1-3 ?g/kg)and a complicated purification scheme,while their biological activities can't be maintained very well.Therefore,we choose the way of gene recombination to express the protein.To avoid serious problems such as a low expression rate,the formation of inclusion bodies,improper protein-folding,and toxicity problems,which may occur during the process of heterologous gene expression and purification in E.coli expression system,eukaryotic cell systems as an alternative describe the production of biologically active rhBMPs through in vitro secreting of Pichia pastoris.The main purpose of the paper was to develop a highly productive and simplified process for rhBMP2 production.For this purpose,the structural analysis of BMP2 protein was carried out firstly,and the gene encoding the propeptied and maturepeptide of BMP2 protein was selected as expressed gene sequence.EcoR I and Not I cleavage sites were designed,in addition the protein purification tag 6×His was added,then the expression plasmids pPIC9K-BMP2 and pPICZaA-BMP2 were successfully constructed.After expression plasmids linearizing,they were transfected into GS115 and X33 strains respectively,and the high-copy strain was induced to express.Afterwards,fermentation products were analyzed by SDS-PAGE and Western blot,and a one-step purifncation scheme was implemented by Ni2+-NTA resin to isolate the biologically active rhBMP2.The rhBMP2 content was determined with Human BMP2 ELISA Kit,and the expression conditions(induction time,methanol concentration,initial bacterial concentration,medium pH)were optimized to determine the optimal expression conditions.Finally,the alkaline phosphatase(ALP)activity and the proliferation-differentiation ability of mouse myoblast(C2C12)cells treated by above rhBMP2 were investigated by alkaline phosphatase kits and MTT method.The calcium deposition was observed by alizarin red staining,and then the cells were identified by morphological analysis.The results showed that GS115/pPIC9K-BMP2 strain,a highly resistant recombinant strain,was successfully screened with 3 mg/mL G418.The optimal conditions were as follows:the initial concentration OD600nm=1.0,the concentration of methanol 1.0%,the induction medium pH6.0,the induction time 72 h,and the highest expression was 189.6 ?g/L.The biological avtivity was the best,and ALP activity was 40.4 U/L in 10 ?g/mL concentration of rhBMP2.