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Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

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Abstract

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-β superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34–38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

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Acknowledgments

We would like to acknowledge the financial support of the Federal Brazilian agencies (FINEP, CNPq, and CAPES) and the São Paulo State Research Foundation (FAPESP) and of the SIN-Implant System Company. We would also like to thank Dr. Inder Verma (The Salk Institute, La Jolla, USA) for kindly supplying the pCMV-IRES-EGFP plasmid, and the excellent technical assistance of Zizi de Mendonça, Débora Cristina da Costa Lopes, Sandra Regina Souza, and Ricardo Krett de Oliveira.

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Authors and Affiliations

  1. NUCEL – Cell and Molecular Therapy Center, University of São Paulo, São Paulo, Brazil

    J. C. Bustos-Valenzuela, E. Halcsik, Ê. J. Bassi, M. A. Demasi, J. M. Granjeiro & M. C. Sogayar

  2. Institute of Chemistry, University of São Paulo, Avenida Professor Lineu Prestes, 748 Bloco 9 sup, São Paulo, SP, 05508-000, Brazil

    J. C. Bustos-Valenzuela, E. Halcsik, M. A. Demasi & M. C. Sogayar

  3. Cell and Molecular Biology Department, Federal Fluminense University, Niterói, RJ, Brazil

    J. M. Granjeiro

Authors
  1. J. C. Bustos-Valenzuela
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  2. E. Halcsik
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  3. Ê. J. Bassi
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  4. M. A. Demasi
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  5. J. M. Granjeiro
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  6. M. C. Sogayar
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Correspondence to M. C. Sogayar.

Additional information

J. C. Bustos-Valenzuela and E. Halcsik equally contributed to this study.

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Bustos-Valenzuela, J.C., Halcsik, E., Bassi, Ê.J. et al. Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells. Mol Biotechnol 46, 118–126 (2010). https://doi.org/10.1007/s12033-010-9287-0

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