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Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays
Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regener...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026339/ https://www.ncbi.nlm.nih.gov/pubmed/32067115 http://dx.doi.org/10.1186/s13568-020-0964-5 |
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author | Suzuki, Miriam Fussae Oliveira, João Ezequiel Damiani, Renata Lima, Eliana Rosa Amaral, Kleicy Cavalcante Santos, Anderson Maikon de Souza Magalhães, Geraldo Santana Faverani, Leonardo Perez Pereira, Luis Antonio Violin Dias Silva, Fabiana Medeiros Bartolini, Paolo |
author_facet | Suzuki, Miriam Fussae Oliveira, João Ezequiel Damiani, Renata Lima, Eliana Rosa Amaral, Kleicy Cavalcante Santos, Anderson Maikon de Souza Magalhães, Geraldo Santana Faverani, Leonardo Perez Pereira, Luis Antonio Violin Dias Silva, Fabiana Medeiros Bartolini, Paolo |
author_sort | Suzuki, Miriam Fussae |
collection | PubMed |
description | Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts. |
format | Online Article Text |
id | pubmed-7026339 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-70263392020-03-02 Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays Suzuki, Miriam Fussae Oliveira, João Ezequiel Damiani, Renata Lima, Eliana Rosa Amaral, Kleicy Cavalcante Santos, Anderson Maikon de Souza Magalhães, Geraldo Santana Faverani, Leonardo Perez Pereira, Luis Antonio Violin Dias Silva, Fabiana Medeiros Bartolini, Paolo AMB Express Original Article Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts. Springer Berlin Heidelberg 2020-02-17 /pmc/articles/PMC7026339/ /pubmed/32067115 http://dx.doi.org/10.1186/s13568-020-0964-5 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Original Article Suzuki, Miriam Fussae Oliveira, João Ezequiel Damiani, Renata Lima, Eliana Rosa Amaral, Kleicy Cavalcante Santos, Anderson Maikon de Souza Magalhães, Geraldo Santana Faverani, Leonardo Perez Pereira, Luis Antonio Violin Dias Silva, Fabiana Medeiros Bartolini, Paolo Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title | Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title_full | Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title_fullStr | Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title_full_unstemmed | Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title_short | Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays |
title_sort | human bone morphogenetic protein-2 (hbmp-2) characterization by physical–chemical, immunological and biological assays |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026339/ https://www.ncbi.nlm.nih.gov/pubmed/32067115 http://dx.doi.org/10.1186/s13568-020-0964-5 |
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