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- Title
Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.
- Authors
Oliveira, João E.; Suzuki, Miriam F.; Damiani, Renata; Lima, Eliana R.; Amaral, Kleicy C.; Santos, Anderson M. S.; Magalhães, Geraldo S.; Faverani, Leonardo P.; Pereira, Luís A. V. D.; Bartolini, Paolo
- Abstract
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.
- Subjects
BONE morphogenetic proteins; AFFINITY chromatography; HIGH performance liquid chromatography; BONE regeneration; PEPTIDES; AUTOTRANSPLANTATION
- Publication
Cells (2073-4409), 2021, Vol 10, Issue 12, p3525
- ISSN
2073-4409
- Publication type
Article
- DOI
10.3390/cells10123525