| dc.description | Human BMP-2, a homodimeric protein that belongs to the TGF- 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. | |
