Expression, Purification And Renaturation Of Recombinant Human Collagen-binding Bone Morphogenetic Protein-2 From Escherichia Coli

Bone morphogenetic protein-2(BMP2), as a member of BMPs, plays an important role in bone regeneration and repair, to control its distribution in vivo and solve the problem about its short half-life and diffusion with the blood, we reformed the natural protein by addinga collagen-binding domain(CBD). Escherichiacoli has several merits, such as the low cost, high-yield, simply manipulation, clear heredity background and so on, it has been used to prepare the recombinant protein commonly. In this article, recombinant human bone BMP2 with CBD was prepared from Escherichia coli. Construction expression, purification, refolding and activity assay of the recombinant protein were studied.Method:The recombinant vector p GEX-6P-1/BMP2, containing GST tag and recombinant human BMP2(rh BMP2)gene segment was construct and transformed into E.coli BL21, the expression of recombinant protein was induced by using isopropyl β-D-thiogalactopyranoside(IPTG) and stirred at 18 ℃. IPTG concentrations were 0.1, 0.2, 0.5 and 1.0 mmol/L, Induction time were 2 h, 4 h and overnight. Another two recombinant vector p ET21b/BMP2, containing 6×His tag and BMP2 gene segment, and recombinant vector p ET21b/CBD-BMP2, containing 6×His tag and CBD-BMP2 gene segment, were construct and transformed into E.coli BL21. The expression of recombinant protein was induced using IPTG(1 mmol) and stirred at 37 ℃, nickel chelate chromatography was used to purify the recombinant monomer. The expression of three recombinant protein were detected by SDS-PAGE method; Dialysis refolding method using different refolding buffers was selected to refold the BMP2 monomer, groups were divided based on different protein concentration before refolding and whether to add L-arginine, the concentration of denaturant was reduced slowly to refold the BMP2 monomer at 4℃;the expression was detected by 15% SDS-PAGE method;The biological activities of CBD-BMP2 was characterized by MC-3T3-E1 cells, incubated the resuscitated MC-3T3-E1 cells at 37 ℃ for 24 h,then inoculated the MC-3T3-E1 cells at 24-hole-board,and each hole has 1×104 cells, added recombinant protein CBD-BMP2 or commercial BMP2 respectively, added 4 different concentrations of refolded BMP2(25 ng/m L、50 ng/m L、75 ng/m L and 100 ng/m L)to incubate with cells for 3 d, the corresponding concentration commercial BMP2 were standard controls, examined the ALP activity of MC-3T3-E1 after incubating for estimating the osteoinducation ability of recombinant BMP2. Then we linked the CBD-BMP2 to the collagen scaffolds and continued incubating at 37 ℃, at various time points(6 h、12 h、24 h、3 d、5 d、7 d and 8 d), the scaffolds was removed and the released protein amount was determined to make the release cure. Compared the effects of different release times on the testing results in order to selete the optimum release time.Results: During the expression progress of BMP2 containing GST purification tag, the concentration of IPTG had less influence on expression quantity, the yield of BMP2 was considerable and the BMP2 showed solubility when induced for 4h, extended the induced time, recombinant protein expressed as inclusion bodies, the expression quantity was much less than that of recombinant protein containing of 6×His purification tag. The recombinant BMP2 containing of 6×His purification tag abundantly expresses as inclusion bodies, we used 8M urea to dissolve the inclusion bodies, the results showed a higher rate of dissolution, after purification using nickel chelate chromatography, the purified recombinant protein BMP2 and CBD-BMP2 existed in elution buffer B with relative molecular mass about 13000; the refolding of BMP2 and CBD-BMP2 performed considerable results when the concentration of recombinant before refolding was lower than 100 μg/m L and the refolding buffer contained L-Arginine, the SDS-PAGE results indicated that the monomer was successfully refolded into dimer with relative molecular mass about 28000. In vitro, the CBD-BMP2 manifested a good but the osteogenic ability of CBD-BMP2 was slightly lower than that of commercial BMP2, the recombinant protein with CBD showed a flat release curve comparing the commercial BMP2 without CBD.Conclusions: recombinant BMP2 consists of GST purification tag expresses a small amount of soluble protein, do not meet the requirements of further experiments; so we apply 6×His purification tag to replace the GST purification tag, recombinant protein abundantly expresses as inclusion bodies, the BMP2 and CBD-BMP2 are refolded by using optimized dilution method, the bioactivity testing experiment schemes of the protein with CBD are compared and determine the best detection method, we use this method to test the biological activity of CBD-BMP2 and prove its succeed in refolding. Comparing with the commercial BMP2, the CBD-BMP2 has a slow release after combined with collagen scaffold.