Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing

Autor(es)

Oliveira, Joao E. ^[1] ; Suzuki, Miriam F. ^[1] ; Damiani, Renata ^[2] ; Lima, Eliana R. ^[2] ; Amaral, Kleicy C. ^[1] ; Santos, Anderson M. S. ^[3] ; Magalhaes, Geraldo S. ^[4] ; Faverani, Leonardo P. ^[3] ; Pereira, Luis A. V. D. ^[5] ; Bartolini, Paolo ^[1]

Afiliação do(s) autor(es)

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

^[2] Biosintesis P&D, BR-05508000 Sao Paulo, SP - Brazil

^[3] UNESP, Sao Paulo State Univ, Sch Dent, Dept Diag & Surg, BR-16015050 Aracatuba, SP - Brazil

^[4] Inst Butantan, Immunopathol Lab, BR-05503900 Sao Paulo, SP - Brazil

^[5] Univ Estadual Campinas, Dept Biochem & Tissue Biol, Inst Biol, State Univ Campinas, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: CELLS; v. 10, n. 12 DEC 2021.
• Citações Web of Science: 0

Resumo

Human BMP-2, a homodimeric protein that belongs to the TGF- beta 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. (AU)

• 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
• Linha de fomento: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE
• 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
• Linha de fomento: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE