KLEICY CAVALCANTE AMARAL
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Artigo IPEN-doc 28508 Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space2021 - OLIVEIRA, JOAO E.; SUZUKI, MIRIAM F.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M.S.; MAGALHAES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; BARTOLINI, PAOLOHuman BMP-2, a homodimeric protein that belongs to the TGF- family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-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.