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

Página do item simplificado
dc.contributor.authorOliveira, João E.
dc.contributor.authorSuzuki, Miriam F.
dc.contributor.authorDamiani, Renata
dc.contributor.authorLima, Eliana R.
dc.contributor.authorAmaral, Kleicy C.
dc.contributor.authorSantos, Anderson M. S. [UNESP]
dc.contributor.authorMagalhães, Geraldo S.
dc.contributor.authorFaverani, Leonardo P. [UNESP]
dc.contributor.authorPereira, Luís A. V. D.
dc.contributor.authorBartolini, Paolo
dc.contributor.institutionIPEN–CNEN
dc.contributor.institutionBiosintesis P & D
dc.contributor.institutionUniversidade Estadual Paulista (UNESP)
dc.contributor.institutionInstituto Butantan
dc.contributor.institutionUniversidade Estadual de Campinas (UNICAMP)
dc.date.accessioned2022-04-28T19:48:27Z
dc.date.available2022-04-28T19:48:27Z
dc.date.issued2021-12-01
dc.description.abstractHuman 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.en
dc.description.affiliationInstituto de Pesquisas Energéticas e Nucleares IPEN–CNEN, Av. Prof. Lineu Prestes 2242
dc.description.affiliationBiosintesis P & D
dc.description.affiliationDepartment of Surgery and Integrated Clinic School of Dentistry Universidade Estadual Paulista Júlio de Mesquita Filho UNESP
dc.description.affiliationImmunopathology Laboratory Instituto Butantan
dc.description.affiliationDepartment of Biochemistry and Tissue Biology Institute of Biology State University of Campinas UNICAMP
dc.description.affiliationUnespDepartment of Surgery and Integrated Clinic School of Dentistry Universidade Estadual Paulista Júlio de Mesquita Filho UNESP
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.description.sponsorshipIdFAPESP: 2015/15446-0
dc.description.sponsorshipIdFAPESP: 2016/24724-6
dc.identifierhttp://dx.doi.org/10.3390/cells10123525
dc.identifier.citationCells, v. 10, n. 12, 2021.
dc.identifier.doi10.3390/cells10123525
dc.identifier.issn2073-4409
dc.identifier.scopus2-s2.0-85121447439
dc.identifier.urihttp://hdl.handle.net/11449/223080
dc.language.isoeng
dc.relation.ispartofCells
dc.sourceScopus
dc.subjectCalvarial critical-size defect
dc.subjectHBMP-2
dc.subjectOsteoinductor
dc.subjectPeriplasmic expression
dc.titleSynthesis of human bone morphogenetic protein-2 (Hbmp-2) in e. coli periplasmic space: its characterization and preclinical testingen
dc.typeArtigo

Arquivos

Coleções

Artigos