Effects of Chemical Additives in Refolding Buffer on Recombinant Human BMP-2 Dimerization and the Bioactivity on SaOS-2 Osteoblasts

Abstract

Bone morphogenetic protein-2 (BMP-2) is a promising osteogenic agent in tissue engineering. BMP-2 is usually expressed in Escherichia coli owing to the high yield and low cost, but the protein is expressed as inclusion bodies. Thus, the bottleneck for BMP-2 production in E. coli is the refolding process. Here, we explored the effects of the refolding buffer composition on BMP-2 refolding. The BMP-2 inclusion body was solubilized in urea and subjected to refolding by the dilution method. Various additives were investigated to improve the BMP-2 refolding yield. Nonreducing SDS-PAGE showed that BMP-2 dimers, the presumably biologically active form, were detected at approximately 25 kDa. The highest yield of the BMP-2 dimers was observed in the refolding buffer that contained ionic detergents (sarkosyl and cetylpyridinium chloride) followed by zwitterionic and nonionic detergents (NDSB-195, NP-40, and Tween 80). In addition, sugars (glucose, sorbitol, and sucrose) in combination with anionic detergents (sodium dodecyl sulfate and sarkosyl) reduced BMP-2 oligomers and increased the BMP-2 dimer yield. Subsequently, the refolded BMP-2s were tested for their bioactivity using the alkaline phosphatase assay in osteogenic cells (SaOS-2), as well as the luciferase reporter assay and the calcium assays. The refolded BMP-2 showed the activities in the calcium deposition assay and the luciferase reporter assay but not in the alkaline phosphatase activity assay or the intracellular calcium assay even though the dimers were clearly detected. Therefore, the detection of the disulfide-linked dimeric BMP-2 in nonreducing SDS-PAGE is an inadequate proxy for the bioactivity of BMP-2.