Human bone morphogenetic protein-2 (hBMP-2) characterization by physical–chemical, immunological and biological assays

• Published in: AMB Express Vol. 10; no. 1; p. 34
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 17.02.2020; Springer Nature B.V; SpringerOpen
• Subjects: Alkaline phosphatase; Bioassays; Biological activity; Biomedical and Life Sciences; Biomedical materials; Biotechnology; BMP-2; Bone growth; Bone morphogenetic protein 2; C2C12 bioassay; Cell culture; CHO cell-derived; Culture media; Dimers; E coli; Efficient osteoinductor; Embryogenesis; Escherichia coli-derived; Extreme values; Gel electrophoresis; Glycosylation; Growth factors; High-performance liquid chromatography; Immunology; Life Sciences; Microbial Genetics and Genomics; Microbiology; Organic chemistry; Original; Original Article; Proteins; Regeneration; Regeneration (physiology); Sodium lauryl sulfate; Transforming growth factor-b; Western blotting; C2C12 bioassay; CHO cell-derived; BMP-2; derived; Efficient osteoinductor; Escherichia coli-derived
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-020-0964-5

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.