Rhbmp-6 Expression, Purification And Identification

Partâ… Expression,purification,and characterization of rhBMP-6Bone morphogenetic proteins(BMPs) belong to the transformation growth factorsβsuperfamily(TGF-β) except BMP-1,which lack the C-terminal conserve domain of TGF-β.BMP-6 is one member of BMPs family and is one of the most potent osteogenesis factors as compared with the other members of BMP family.BMP-6 is induced in different stages of bone fracture.BMP-6 promotes bone regeneration in different bone deficiency model.In addition,BMP-6 administered systematically, restores bone volume and micro-architecture in osteoporotic rat and mouse.These results indicate BMP-6 as a therapeutic potential candidate for treating bone deficiency and osteoporosis.In the present study,we recombinant produced BMP-6 in Escherichia coli and cultivated mammalian cells.1.Expression,refolding,and purification of BMP-6 in E.ColiThe variants of BMP-6 and BMP-6 mutant(Ala⁵⁶-His) plus an N-terminal extension(MA or MG duo to the clone site) were expressed in E.Coli as non-fusion protein,respectively.The expression level of the mature peptide with MA extension (MA-BMP-6(1323)) was significantly higher than the other's by screening of host cells and expression constructs under different growth conditions.This construct was transformed into an E.coli BL21(DE3) for the expression of BMP-6 mature peptide.BMP-6 was expressed as IBs after induction,contributing up to 20%of the total bacterial protein.Protein was near homogenous after a simplified procedure of IBs isolation.The IBs were dissolved in 6M Gdn-HCl and 0.1M DTT containing buffer. The refolded dimeric protein was obtained by dilution the dissolved IBs protein in an optimized refolding buffer and about 10%of BMP-6 formed disulphide-linked homodimers.This buffer system was optimized step by step,including solubility, refolding,and refolding adding agent determined.BMP-6 mutant could not improve efficiency of refolding.The refolded dimeric BMP-6 was purified by one step reverse phase liquid chromatography.Howerver,the dose-dependent alkaline phosphatase (ALP) induction activity of the purified dimeric protein was not observed,probably due to lack of N-glycosylation produced in E.Coli.2.Expression,purification,and characterization of BMP-6 in CHO cellsThe prepromature peptide of BMP-6 and prepro-peptide of BMP-2 fused with mature peptide of BMP6(BMP2/6) was subcloned into the mammalian expression vector,respectively.The BMP2/6 construction could efficiently pruduce BMP-6 protein after transient transfection in Cos 7 via liposame reagent.BMP-6 was stable epressed in dihydroflolate reductase deficiency Chinese hamster ovary(CHO-dhfr-) cells.BMP-6 expression levels of the most productive subclones was about 20 ng/10⁶ cells/24 h after individual and cell pool based selection followed by target gene amplification.The specific growth rate,BMP-6 production, and stability of BMP-6 in culture condition and conditioned medium were characterized.BMP-6 was purified by combination of heparin affinity and reverse phase chromatography.The purified product was indentified by SDS-PAGE and western blot.Finally,the purified BMP-6 induced alkaline phophatase(ALP) activity in a time-course and dose-dependent manner.The ALP activity was significantly induced at concentration of 25ng/ml.EC50 was about 100-150ng/ml and the maximal effective dose was about 400ng/ml.The ALP activity was significantly induced after 3 days incubation at concentration of 50ng/ml.In summary,the non-fusion human BMP-6 was expressed in E.coli as IBs with high level.After the optimized procedure for IBs isolation and refolding,the refolded dimeric protein was purified by one step reverse phase liquid chromatography. Furthermore,the BMP-6 was expressed in CHO cells.After vector construction, stable cell line screening,and purification scheme optimazation,the bioactive BMP-6 protein was produced.Partâ…¡Effect and mechanism of BMP-6 on fibrosis and oxidant stressBMP-6 plays a role during embryonal development and tissue morphogenesis postnatal as well as bone formation.Downregulation of BMP-6 may predispose to renal deficiency and chronic renal deficiency directly reduced bone formation, indicating that suppression of chronic renal deficiency my improve bone regeneration. In the present study,the effect and mechamism of BMP-6 on renal fibrosis and oxidant injury were investigated.1.BMP-6 reverses TGF-β1-initiated changes of PTECTransforming growth factor-beta 1(TGF-β1) plays a crucial role in the inception and progression of renal tubulointerstitial fibrosis which lead to renal deficiency. Treatment with TGF-β1 reduced cell proliferation,induced epithelial-to-mesenchymal transition(EMT),deceased expression and activity of matrix metalloproteinases 2(MMP-2),increased expression of fibronectin and tissue inhibitors of matrix metalloproteinases 2(TIMP-2),and increased cell adhesion in renal proximal tubular epithelial cell(PTEC) HK-2 cells.All these effects were inhibited when TGF-β1 was combined with BMP-6,whereas BMP-6 alone had no such effects.In addition,BMP-6 abrogated the JNK and Smad3 signaling activated by TGF-β1.BMP6 ameliorated TGF-β1 induced fibrosis changes in HK-2 cells and suppression of TGF-β1-mediated JNK and Smad3 activation maybe implicated.2.BMP-6 attenuates oxidant injury in PTEC via Smad-dependent HO-1 inductionReactive oxygen species are involved in TGF-β1-induced transition and heme oxygenase 1(HO-1) induction is a protective response to oxidative stress.BMP-6 effectively protected renal proximal tubule cells(HK-2) against hydrogen peroxide (H₂O₂)-induced cell injury.BMP-6 also increased HO-1 gene expression.Inhibition of de novo gene expression,HO-1 inhibitor,HO-1 knockdown,or carbon monoxide (CO) scavenger attenuated the cytoprotective effect of BMP-6,whereas HO-1 inducer, HO-1 constitutive expression,or HO-1 products bilirubin and CO ameliorated H₂O₂-induced cell injury.Stimulation of HK-2 cells with BMP-6 activated Smad5 signaling and BMP6-mediated induction of HO-1 expression was inhibited by Smad5 knockdown.Furthermore,deletion or mutation of the Smad-binding element in HO-1 promoter inhibited the BMP-6-induced HO-1 promoter activity.These findings suggest that induction of HO-1 through a Smad-dependent manner is responsible for the cytoprotective effect of BMP-6 in H₂O₂-mediated renal cell injury.In summery,the antifibrotic and antioxidant effect of BMP-6 were determined in renal proximal tubular epithelial cells,useful for further therapeutic investigations of BMP-6 in the treatment of renal interstitial fibrosis and bone deficiency.