KLEICY CAVALCANTE AMARAL
2 resultados
Resultados de Busca
Agora exibindo 1 - 2 de 2
Artigo IPEN-doc 26665 Human bone morphogenetic protein‑2 (hBMP‑2) characterization by physical–chemical, immunological and biological assays2020 - SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M. de S.; MAGALHÃES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; SILVA, FABIANA M.; BARTOLINI, PAOLOCommercially 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, sizeexclusion 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 methBMP- 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.Resumo IPEN-doc 26740 Human bone morphogenetic protein (hBMP)-2 characterization by physical chemical, immunological and biological assays2019 - SUZUKI, M.F.; OLIVEIRA, J.E.; DAMIANI, R.; LIMA, E.R.; AMARAL, K.C.; SILVA, F.M.; BARTOLINI, P.Commercial preparations of human-met-BMP-2 (GenScript) and of CHO-derived hBMP-2 (Infuse-Medtronic) provided a complete characterization of this protein, which belongs to the “transforming growth factors β” superfamily, via SDS-PAGE, Western blotting, reversed-phase HPLC, high-performance size-exclusion chromatography and MALDI-TOF-MS. E.coli-derived met-hBMP-2 has shown a large presence of dimer (MM= 26,054 Da), versus a theoretic value of 26,072 Da. More complex was the distribution of the CHO-derived product, whose exact MM has never been reported due to variable glycosylation: via MALDI-TOF-MS a dimer (28,732 Da) and a large amount of monomer (14,377 Da) were found. A novel method based on RP-HPLC was also validated for hBMP-2 qualitative and quantitative analysis directly in ongoing culture media. The classical “in vitro” bioassay, via alkaline phosphatase induction in murine myoblastic cells C2C12, confirmed that hBMP-2 bioactivity is mostly related to the dimer, being ∼6-fold higher for the CHO-derived glycosylated form. Considering that hBMP-2 is a highly effective osteoinductors, plays an important role during bone regeneration and repair, as well as during embryonic development, and presents an extremely high aggregate value, we believe that these data pave the way to the characterization of this important factor when obtained by DNA recombinant techniques in different host cells.