title: On-column refolding of bone morphogenetic protein-2 using cation exchange resin.
abstract: Refolding is often the bottle-neck step in producing recombinant proteins from inclusion bodies of Escherichia coli, especially for dimer proteins. The refolding process is protein specific, engaging a lot of time and cost to optimize conditions so that the thermodynamics favor protein refolding over competitive aggregation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic agent having significant applications in bone regeneration therapy. In this study, we present a novel solid-phase refolding method for rapid and efficient refolding of recombinant BMP-2 dimer from E. coli. We employed a weak cation exchange resin as the adsorbing support, with decreasing gradient of denaturing agent and exposure to oxidizing conditions for adequate disulfide bond formation. Refolded BMP-2 was further purified using size exclusion chromatography and analyzed for its secondary structure and biological activity. The purified BMP-2 dimer showed dose-dependent induction of alkaline phosphatase (ALP) activity in MC3T3 pre-osteoblast cells, thus translating the success of our refolding method. This simple and rapid method can also be applied in refolding and purification of other BMP-2 like dimer proteins.
authors.affiliation: University of the Sciences, Department of Pharmaceutical Sciences, Philadelphia, PA 19104, USA.
authors.forename: Anuja M
authors.initials: AM
authors.lastname: Rane
authors.forename: Sriramakamal
authors.initials: S
authors.lastname: Jonnalagadda
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Li
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry* / Q000494:pharmacology; D002411:Cation Exchange Resins / Q000737:chemistry*; D002460:Cell Line; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D002411:Cation Exchange Resins; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase
keywords: Bone morphogenetic protein-2; Cation exchange chromatography; Inclusion body; On-column refolding; Protein dimer