Proteína morfogenética de hueso 2: expresión heteróloga y potencial en la regeneración ósea

Andrea Mesa-Restrepo; Juan Fernando-Alzate; Edwin Bairon Patiño-Gonzalez

doi:10.17533/udea.acbi.v43n114a01

Autores

Andrea Mesa-Restrepo Universidad de Antioquia, Medellin, Colombia https://orcid.org/0000-0001-7324-2105
Juan Fernando-Alzate Universidad de Antioquia, Medellin, Colombia
Edwin Bairon Patiño-Gonzalez Universidad de Antioquia, Medellin, Colombia https://orcid.org/0000-0003-0958-9176

DOI:

https://doi.org/10.17533/udea.acbi.v43n114a01

Palavras-chave:

Células C2C12, Fosfatasa alcalina, Renaturalización, hBMP‐2

Resumo

Atualmente, a proteína morfogênica óssea 2 (BMP-2) é um dos dois fatores de crescimento mais osteoindutores usados clinicamente em dispositivos médicos que promovem a formação óssea. Normalmente esta proteína é comprada a preços elevados e em pequenas quantidades que não são suficientes para cobrir as necessidades clínicas. Por isso, tem sido proposto que os centros de pesquisa utilizem seus próprios sistemas heterólogos para obter um abastecimento constante. O objetivo deste trabalho é padronizar a expressão heteróloga de BMP-2 e avaliar sua atividade osteoindutora in vitro. Nosso procedimento para expressão e purificação foi baseado na tecnologia de DNA recombinante usando o plasmídeo pET-28 e IPTG como indutor. Depois de extrair a proteína dos corpos de inclusão, renaturá-la e modificá-la usando um sistema redox, um dímero de 26 kDa foi observado por eletroforese. Sua atividade osteoindutiva foi avaliada em células mioblásticas C2C12 por quantificação enzimática da atividade da fosfatase alcalina (ALP) e coloração dos nódulos de mineralização. A atividade da fosfatase alcalina foi proporcional à concentração de BMP-2, obtendo-se um aumento de 90% nas concentrações de 3 µg/mL. Essas células formaram nódulos de cálcio, mineralizando a superfície em 50%.

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Biografia do Autor

Andrea Mesa-Restrepo, Universidad de Antioquia, Medellin, Colombia

Grupo de Biomateriales Avanzados y Medicina Regenerativa, Universidad de Antioquia, Medellín, Colombia.

Juan Fernando-Alzate, Universidad de Antioquia, Medellin, Colombia

Centro Nacional de Secuenciación Genómica, Universidad de Antioquia, Medellín, Colombia.

Edwin Bairon Patiño-Gonzalez, Universidad de Antioquia, Medellin, Colombia

Grupo Bioquímica Estructural de Macromoléculas, Universidad de Antioquia, Medellín, Colombia

Referências

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