Bone morphogenetic protein 2: heterologous expression and potential in bone regeneration
DOI:
https://doi.org/10.17533/udea.acbi.v43n114a01Keywords:
Alkaline phosphatase, Cells C2C12, Refolding protein, hBMP‐2Abstract
Currently, bone morphogenetic protein 2 (BMP-2) is one of the two osteoinductive growth factors used in medical devices to promote bone formation. Typically, this protein is bought from commercial houses at high rates and in small quantities that are not enough to cover clinical needs. Because of this, it has been proposed that research centers use their own heterologous expression systems to have a constant supply of BMP-2. The aim of this study was to standardize the heterologous expression of BMP-2 and evaluate its osteoinductive activity in vitro. Our procedure for expression and purification was based on recombinant DNA technology using the plasmid pET-28 and IPTG as inductor. After extracting the protein from inclusion bodies, folding it and modifying it via a redox system, we observed via electrophoresis a 26 kDa dimer. We evaluated its osteoinductive activity in myoblastic C2C12 by quantifying enzymatically the activity of alkaline phosphate (ALP) and staining mineralization nodules. ALP activity is proportional to BMP-2 concentration, increasing 90% at 3 µg/mL. These cells form calcium nodules, mineralizing 50% of the area.
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Allendorph, G.P., Vale, W.W., & Choe, S. (2006). Structure of the ternary signaling complex of a TGF-β superfamily member. Proceedings of the National Academy of Sciences of the United States of America, 103(20), 7643-7648. DOI:10.1073/pnas.0602558103
Bahney, C.S., Zondervan, R.L., Allison, P., Theologis, A., Ashley, J.W., Ahn, J., Miclau, T., Marcucio, R.S., & Hankenson, K.D. (2019). Cellular biology of fracture healing. Journal of Orthopaedic Research, 37(1), 35–50. DOI:10.1002/jor.24170
Basak, S., Paul Nobrega, R., Tavella, D., Deveau, L. M., Koga, N., Tatsumi-Koga, R., Baker, D., Massi, F., & Robert Matthews, C. (2019). Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein. Proceedings of the National Academy of Sciences of the United States of America, 116(14), 6806–6811. DOI:10.1073/pnas.1818744116
DeLano, W.L. (2002). PyMOL: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography, 40, 82–92. http://148.79.162.84/newsletters/newsletter40/11_pymol.pdf
Durham, E.L., Howie, R.N., Hall, S., Larson, N., Oakes, B., Houck, R., Grey, Z., Steed, M., Larue, A.C., Muise-Helmericks, R., & Cray, J. (2018). Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold 11 Medical and Health Sciences 1103 Clinical Sciences 09 Engineering 0903 Biomedical Engineering. Journal of Translational Medicine, 16(1), 1–8. DOI:10.1186/s12967-018-1697-y
Englander, S.W., & Mayne, L. (2014). The nature of protein folding pathways. Proceedings of the National Academy of Sciences of the United States of America, 111(45), 15873–15880. DOI:10.1073/pnas.1411798111
Fung, S.L., Wu, X., Maceren, J.P., Mao, Y., & Kohn, J. (2019). In vitro evaluation of recombinant bone morphogenetic protein-2 bioactivity for regenerative medicine. Tissue Engineering - Part C: Methods, 25(9), 553–559. DOI:10.1089/ten.tec.2019.0156
Hettiaratchi, M.H., Krishnan, L., Rouse, T., Chou, C., McDevitt, T.C., & Guldberg, R.E. (2020). Heparin-mediated delivery of bone morphogenetic protein-2 improves spatial localization of bone regeneration. Science Advances, 6(1), 1–12. DOI:10.1126/sciadv.aay1240
Ihm, H.J., Yang, S.-J., Huh, J.-W., Choi, S.Y., & Cho, S.-W. (2008). Soluble expression and purification of synthetic human bone morphogenetic protein-2 in Escherichia coli. BMB Reports, 41(5), 404–407. DOI:10.5483/BMBRep.2008.41.5.404
Kashiwagi, K., Tsuji, T., & Shiba, K. (2009). Directional BMP-2 for functionalization of titanium surfaces. Biomaterials, 30(6), 1166–1175. DOI:10.1016/j.biomaterials.2008.10.040
Kim, H.S., Neugebauer, J., McKnite, A., Tilak, A., & Christian, J.L. (2019). BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis. ELife, 8, 1–22. DOI:10.7554/eLife.48872
Krauss, I.R., Merlino, A., Vergara, A., & Sica, F. (2013). An overview of biological macromolecule crystallization. International Journal of Molecular Sciences, 14(6), 11643–11691. DOI:10.3390/ijms140611643
Krishnakumar, G.S., Roffi, A., Reale, D., Kon, E., & Filardo, G. (2017). Clinical application of bone morphogenetic proteins for bone healing: a systematic review. International Orthopedics, 41, 1073–1083. DOI:10.1007/s00264-017-3471-9
Long, S., Truong, L., Bennett, K., Phillips, A., Wong-Staal, F., & Ma, H. (2006). Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli. Protein Expression and Purification, 46(2), 374-378. DOI:10.1016/j.pep.2005.09.025
Mevel, R., Draper, J.E., Lie-A-Ling, M., Kouskoff, V., & Lacaud, G. (2019). RUNX transcription factors: Orchestrators of development. Development (Cambridge), 146, 1–19. DOI:10.1242/dev.148296
Miyazono, K., Kamiya, Y., & Morikawa, M. (2010). Bone morphogenetic protein receptors and signal transduction. Journal of Biochemistry, 147(1), 35–51. DOI:10.1093/jb/mvp148
Mueller, T.D., & Nickel, J. (2012). Promiscuity and specificity in BMP receptor activation. FEBS Letters, 586(14), 1846–1859. DOI:10.1016/j.febslet.2012.02.043
Nickel, J., & Mueller, T.D. (2019). Specification of BMP Signaling. Cells, 8(12), 1579. DOI:10.3390/cells8121579
Park, S.Y., Kim, K.H., Kim, S., Lee, Y.M., & Seol, Y.J. (2019). BMP-2 gene delivery-based bone regeneration in dentistry. Pharmaceutics, 11(8), 1–23. DOI:10.3390/pharmaceutics11080393
Rahman, M.S., Akhtar, N., Jamil, H.M., Banik, R.S., & Asaduzzaman, S.M. (2015). TGF-β/BMP signaling and other molecular events: Regulation of osteoblastogenesis and bone formation. Bone Research, 3, Article 15005. DOI:10.1038/boneres.2015.5
Ruehle, M.A., Krishnan, L., Vantucci, C.E., Wang, Y., Stevens, H.Y., Roy, K., Guldberg, R.E., & Willett, N.J. (2019). Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss. Journal of Orthopaedic Research, 37(3), 553–561. DOI:10.1002/jor.24225
Ruppert, R., Hoffmann, E., & Sebald, W. (1996). Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. European Journal of Biochemistry, 237(1), 295–302. DOI:10.1111/j.1432-1033.1996.0295n.x
Saraff, V., Narayanan, V.K., Lawson, A.J., Shaw, N.J., Preece, M.A., & Högler, W. (2016). A diagnostic algorithm for children with low alkaline phosphatase activities: Lessons learned from laboratory screening for hypophosphatasia. Journal of Pediatrics, 172, 181-186e1. DOI:10.1016/j.jpeds.2016.01.045
Scheufler, C., Sebald, W., Hulsmeyer, M., Hu, M., & Hülsmeyer, M. (1999). Crystal Structure of Human Bone Morphogenetic Protein-2 at 2.7 A Resolution. Journal of Molecular Biology, 287, 103–115. DOI:10.1006/jmbi.1999.2590
Sharapova, N.E., Kotnova, A.P., Galushkina, Z.M., Lavrova, N.V., Poletaeva, N.N., Tukhvatulin, A.E., Semikhin, A.S., Gromov, A.V., Soboleva, L.A., Ershova, A.S., Zaitsev, V.V., Sergienko, O.V., Lunin, V.G., & Karyagina, A.S. (2010). Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo. Molecular Biology, 44(6), 923–930. DOI:10.1134/S0026893310060099
Tabisz, B., Schmitz, W., Schmitz, M., Luehmann, T., Heusler, E., Rybak, J., Meinel, L., Fiebig, J.E., Mueller, T.D., & Nickel, J. (2017). Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Sca ff olds. Biomacromolecules, 18(3):695-708. DOI:10.1021/acs.biomac.6b01407
Uday, S., Matsumura, T., Saraff, V., Saito, S., Orimo, H., & Högler, W. (2019). Tissue non-specific alkaline phosphatase activity and mineralization capacity of bi-allelic mutations from severe perinatal and asymptomatic hypophosphatasia phenotypes: Results from an in vitro mutagenesis model. Bone, 127(January), 9–16. DOI:10.1016/j.bone.2019.05.031
Vallejo, L.F., Brokelmann, M., Marten, S., Trappe, S., Cabrera-Crespo, J., Andrea Hoffmann, A., Gross, G., Weich, H.A., & Rinas, U. (2002). Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. Journal of Biotechnology, 94 (2), 185-194. DOI:10.1016/S0168-1656(01)00425-4.
Von Einem, S., Schwarz, E., & Rudolph, R. (2010). A novel TWO-STEP renaturation procedure for efficient production of recombinant BMP-2. Protein Expression and Purification, 73(1), 65-69. DOI:10.1016/j.pep.2010.03.009
Xiong, S., Zhang, L., & He, Q.-Y. (2008). Fractionation of proteins by heparin chromatography. Methods in Molecular Biology (Clifton, N.J.), 424, 213–221. DOI:10.1007/978-1-60327-064-9_18
Yilgor, P., Tuzlakoglu, K., Reis, R. L., Hasirci, N., & Hasirci, V. (2009). Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering. Biomaterials, 30(21), 3551–3559. DOI:10.1016/j.biomaterials.2009.03.024
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