en 30555651 <p>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 <em>Escherichia coli (E. coli)</em> 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.<br /> 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 <em>E. coli </em>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.<br /> 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.<br /> Conclusion: A new method for production of biologically active dimeric form of BMP-2 in <em>E. coli </em>expression system was established in this study.</p> Bone morphogenetic protein -2, Cloning, Escherichia coli, Inclusion bodies, Protein refolding 202 207 https://www.ajmb.org/En/Article.aspx?id=326 https://www.ajmb.org/PDF/En/FullText/326.pdf DavoodNasrabadiStudent Research Committee, Semnan University of Medical Sciences, Semnan, Iran1310SiamakRezaeianiDepartment of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran1311AliSayadmaneshDepartment of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran1312MohammadrezaBaghaban EslaminejadDepartment of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran1313AliakbarShabaniDepartment of Medical Biotechnology, Semnan University of Medical Sciences, Semnan, Iran1314