title: Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing.
abstract: One problem associated with clinical application of CHO-derived recombinant human bone morphogenetic protein (C-BMP-2) is its high cost due to the need for use of high doses. To solve this problem, Escherichia coli-derived BMP-2 (E-BMP-2) has been examined using the technique of molecular unfolding and refolding. However, it is unclear whether the characteristics of E-BMP-2 are appropriate for clinical application. In this study, we examined the biological activity of E-BMP-2 and its heat tolerance in in vitro and in vivo systems. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the high purity of E-BMP-2. E-BMP-2-induced alkaline phosphatase expression in osteoprogenitor cells (C2C12, ST2, and primary murine calvarial osteoblast cells) was dose-dependent, and consistently elicited ectopic new ossicles of significant size in mice, also in dose-dependent fashion. In addition, E-BMP-2 induced phosphorylation of Smad1/5/8 and mRNA expression of osteoblastic differentiation markers to the same extent as C-BMP-2. On the other hand, when E-BMP-2 was exposed to increasing heat over time, its bone-inducing capacity was maintained until reaching 70 degrees C for 2 h or 90 degrees C for 15 min. Thus, E-BMP-2 will exhibit a decrease in activity with the sterilization procedures required prior to use in surgery. These findings indicate that the biological capacity and heat stability of E-BMP-2 are almost equivalent to those of currently available C-BMP-2, and suggest that E-BMP-2 might, thus, solve current problems of cost impeding routine clinical use of rhBMP-2.
authors.affiliation: Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Yano
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Hoshino
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ohta
authors.forename: Tomoya
authors.initials: T
authors.lastname: Manaka
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Naka
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Imai
authors.forename: Walter
authors.initials: W
authors.lastname: Sebald
authors.forename: Kunio
authors.initials: K
authors.lastname: Takaoka
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D001669:Biochemical Phenomena / Q000187:drug effects*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D001842:Bone and Bones / Q000000981:diagnostic imaging / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010012:Osteogenesis / Q000187:drug effects*; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects*; D011859:Radiography; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D051785:Smad Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000494:pharmacology*
chemical_list: D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D000469:Alkaline Phosphatase