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Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing

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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°C for 2 h or 90°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.

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Acknowledgments

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Project Grants 16109009 and 1679085 for KT, and 19791018 for YI).

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan

    Koichi Yano, Masatoshi Hoshino, Yoichi Ohta, Tomoya Manaka, Yoshifumi Naka, Yuuki Imai & Kunio Takaoka

  2. Physiological Chemistry II, Theodor-Boveri-Institute for Biocenter of Würzburg University, Würzburg, Germany

    Walter Sebald

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  1. Koichi Yano
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  2. Masatoshi Hoshino
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  3. Yoichi Ohta
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  4. Tomoya Manaka
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  5. Yoshifumi Naka
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  6. Yuuki Imai
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  7. Walter Sebald
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  8. Kunio Takaoka
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Correspondence to Yuuki Imai.

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Yano, K., Hoshino, M., Ohta, Y. et al. Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing. J Bone Miner Metab 27, 355–363 (2009). https://doi.org/10.1007/s00774-009-0040-3

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  • DOI: https://doi.org/10.1007/s00774-009-0040-3

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