Recombinant Human Bone Morphogenetic Protein-2: Optimization of Overproduction, Solubilization, Renaturation and Its Characterization

A codon-optimized synthetic gene encoding recombinant human Bone Morphogenetic Protein 2 (rhBMP-2) fused to thioredoxin-6x-histidine tag at its amino terminus was previously constructed and the recombinant product as a monomer expressed in Escherichia coli BL21(DE3) was confirmed by nano-LC mass spectrometry (LC-MS/MS2) analysis. In this study, we optimized the conditions for overproduction, solubilization of Inclusion Bodies (IB) and dimerization of rhBMP-2 monomer. Overproduction was optimized at various isopropyl-β-D-thiogalactopyranoside concentrations and incubation temperatures. Different kinds of buffer at pH 8.0 to 9.0 were applied for optimization of solubilization and dimerization. Enterokinase cleavage, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western Blot analyses were applied in protein characterization. The activity of rhBMP-2 dimer was determined by production of alkaline phosphatase on C2C12 cells. Under the optimal condition found in this study, 11.5 g cell wet weight or 0.6 g rhBMP-2 monomer per L culture was produced. The soluble monomer of 64 mg was resulted from 200 mg of IB using buffer containing 4 M urea, 0.1 M NaCl, 0.02 M tris-HCl, pH 8.0, 5 mM EDTA and 5 mM dithiothreitol. The dimer to monomer ratio of 4:5 was resulted from dimerization using buffer containing 4 M urea, 100 mM tris-HCl, 5 mM EDTA. The rhBMP-2 cleaved by enterokinase gave correct protein fragments, was recognized by monoclonal antibody against rhBMP-2 and the protein was proved to be biologically active. In conclusion, this study we provide evidence that after optimized solubilization and renaturation steps, rhBMP-2 fusion protein expressed from synthetic gene was biologically active.