Synthesis of human bone morphogenetic protein-2 (Hbmp-2) in e. coli periplasmic space: its characterization and preclinical testing

• Autor(a) principal: Oliveira, João E.
• Data de Publicação: 2021
• Outros Autores: Suzuki, Miriam F., Damiani, Renata, Lima, Eliana R., Amaral, Kleicy C., Santos, Anderson M. S. [UNESP], Magalhães, Geraldo S., Faverani, Leonardo P. [UNESP], Pereira, Luís A. V. D., Bartolini, Paolo
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://dx.doi.org/10.3390/cells10123525; http://hdl.handle.net/11449/223080
• topic: Calvarial critical-size defect; HBMP-2; Osteoinductor; Periplasmic expression

Human BMP-2, a homodimeric protein that belongs to the TGF-β 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.