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

Autor(es):Mostrar menos -

Suzuki, Miriam Fussae ^[1] ; Oliveira, Joao Ezequiel ^[1] ; Damiani, Renata ^[2] ; Lima, Eliana Rosa ^[1] ; Amaral, Kleicy Cavalcante ^[1] ; Santos, Anderson Maikon de Souza ^{[3, 4]} ; Magalhaes, Geraldo Santana ^[5] ; Faverani, Leonardo Perez ^{[3, 4]} ; Pereira, Luis Antonio Violin Dias ^[6] ; Silva, Fabiana Medeiros ^[2] ; Bartolini, Paolo ^[1]

Afiliação do(s) autor(es)

^[1] IPEN CNEN SP, Inst Pesquisas Energet & Nucl, Ctr Biotechnol, Ave Prof Lineu Prestes 2242, BR-05508000 Sao Paulo, SP - Brazil

^[2] Biosintesis P&D, Sao Paulo, SP - Brazil

^[3] Univ Estadual Paulista Julio de Mesquita Filho UN, Sch Dent, Dept Surg, Aracatuba, SP - Brazil

^[4] Univ Estadual Paulista Julio de Mesquita Filho UN, Sch Dent, Integrated Clin, Aracatuba, SP - Brazil

^[5] Inst Butantan, Immunopathol Lab, Sao Paulo, SP - Brazil

^[6] Univ Estadual Campinas UNICAMP, Inst Biol, Dept Biochem & Tissue Biol, Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: AMB EXPRESS; v. 10, n. 1 FEB 17 2020.
• Citações Web of Science: 0

Resumo

Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor beta (TGF-beta) 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. (AU)

• Processo FAPESP: 16/24724-6 - Expressão, purificação e caracterização da proteína morfogenética humana (BMP-2) para aplicação em biomateriais compósitos com o polímero poli(e-caprolactona)
• Beneficiário: Paolo Bartolini
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE
• Processo FAPESP: 15/15446-0 - Expressão, purificação e caracterização da proteína morfogenética humana (BMP-2) para aplicação em biomateriais compósitos com o polímero poli(e-caprolactona)
• Beneficiário: Paolo Bartolini
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE