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Title

INCLUSION BODY EXPRESSION AND REFOLDING OF RECOMBINANT BONE MORPHOGENETIC PROTEIN-2

Writers

NASRABADI DAVOOD | REZAEIANI SIAMAK | SAYADMANESH ALI | BAGHABAN ESLAMINEJAD MOHAMADREZA | SHABANI ALIAKBAR

Pages

 Start Page 202 | End Page 207

Abstract

 Background: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes ESCHERICHIA COLI (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems.Methods: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in Origami TM E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line.Results: No difference was observed between oxidoreductase systems in improving the efficiency of PROTEIN REFOLDING. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein.Conclusion: A new method for production of biologically active dimeric form of BMP-2 inE. coli expression system was established in this study.

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