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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
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]
Total Authors: 10
Affiliation:
[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
Total Affiliations: 5
Document type: Journal article
Source: CELLS; v. 10, n. 12 DEC 2021.
Web of Science Citations: 0
Abstract

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)

FAPESP's process: 15/15446-0 - Expression, purification and characterization of bone morphogenetic protein (BMP-2) for application in composites biomaterials with poly(E-caprolactone)
Grantee:Paolo Bartolini
Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
FAPESP's process: 16/24724-6 - Expression, purification and characterization of bone morphogenetic protein (BMP-2) for application in composites biomaterials with poly(E-caprolactone)
Grantee:Paolo Bartolini
Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE