Oliveira, Joao E.
Suzuki, Miriam F.
Lima, Eliana R.
Amaral, Kleicy C.
Santos, Anderson M. S.
Magalhaes, Geraldo S.
Faverani, Leonardo P.
Pereira, Luis A. V. D.
Total Authors: 10
 IPEN CNEN, Inst Pesquisas Energet & Nucl, Ave Prof Lineu Prestes 2242, BR-05508000 Sao Paulo, SP - Brazil
 Biosintesis P&D, BR-05508000 Sao Paulo, SP - Brazil
 UNESP, Sao Paulo State Univ, Sch Dent, Dept Diag & Surg, BR-16015050 Aracatuba, SP - Brazil
 Inst Butantan, Immunopathol Lab, BR-05503900 Sao Paulo, SP - Brazil
 Univ Estadual Campinas, Dept Biochem & Tissue Biol, Inst Biol, State Univ Campinas, BR-13083970 Campinas, SP - Brazil
Total Affiliations: 5
Web of Science Citations:
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)