| Autor: |
João E. Oliveira, Miriam F. Suzuki, Renata Damiani, Eliana R. Lima, Kleicy C. Amaral, Anderson M. S. Santos, Geraldo S. Magalhães, Leonardo P. Faverani, Luís A. V. D. Pereira, Paolo Bartolini |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Cells, Vol 10, Iss 12, p 3525 (2021) |
| Druh dokumentu: |
article |
| ISSN: |
2073-4409 |
| DOI: |
10.3390/cells10123525 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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