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Recombinant human Bone Morphogenetic Protein-2: Optimization of overproduction, solubilization, renaturation and its characterization
Pramesti H.T.a,b, Suciati T.a, Indrayati A.a, Asjarie S.a, Retnoningrum D.S.a
a School of Pharmacy, Institut Teknologi Bandung, Indonesia
b Department of Oral Biology, Faculty of Dentistry, Universitas Padjadjaran, Indonesia
[vc_row][vc_column][vc_row_inner][vc_column_inner][vc_separator css=”.vc_custom_1624529070653{padding-top: 30px !important;padding-bottom: 30px !important;}”][/vc_column_inner][/vc_row_inner][vc_row_inner layout=”boxed”][vc_column_inner width=”3/4″ css=”.vc_custom_1624695412187{border-right-width: 1px !important;border-right-color: #dddddd !important;border-right-style: solid !important;border-radius: 1px !important;}”][vc_empty_space][megatron_heading title=”Abstract” size=”size-sm” text_align=”text-left”][vc_column_text]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 MNaCl, 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. © 2012 Asian Network for Scientific Information.[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Author keywords” size=”size-sm” text_align=”text-left”][vc_column_text]ALkaline phosphatase,Amino terminus,Bone-Morphogenetic Protein-2,C2C12 cells,Dithiothreitol,Fusion proteins,Human bone morphogenetic protein,Human bones,Incubation temperatures,Monomer ratio,Nano-LC,Optimal conditions,Protein characterization,Protein fragments,Renaturation,rhBMP-2,Solubilization,Solubilization of inclusion bodies,Sulphate-polyacrylamide gel electrophoresis,Synthetic genes,Western-blot analysis,Wet weight[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Indexed keywords” size=”size-sm” text_align=”text-left”][vc_column_text]Bone-Morphogenetic Protein-2,Dimerization,Fusion protein,Solubilization[/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”Funding details” size=”size-sm” text_align=”text-left”][vc_column_text][/vc_column_text][vc_empty_space][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][vc_empty_space][megatron_heading title=”DOI” size=”size-sm” text_align=”text-left”][vc_column_text]https://doi.org/10.3923/biotech.2012.133.143[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/4″][vc_column_text]Widget Plumx[/vc_column_text][/vc_column_inner][/vc_row_inner][/vc_column][/vc_row][vc_row][vc_column][vc_separator css=”.vc_custom_1624528584150{padding-top: 25px !important;padding-bottom: 25px !important;}”][/vc_column][/vc_row]