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


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Article
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Portuguese
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Open access
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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.
Reference
Oliveira JE., Suzuki MF., Damiani R, Lima ER., Amaral KC., Santos AM.S., et al. Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing. Cells. 2021 Dec; 10(12): 3525. doi:10.3390/cells10123525.
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https://repositorio.butantan.gov.br/handle/butantan/4086
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Issue Date
2021


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