재조합 hBMP-2의 발현과 rhBMP-2의 생물학적 활성 연구

Abstract

ABSTRACT

Study on the expression of recombinant bone morphogenetic protein-2 and its biological activity

Shin, Hee June Advisor : Prof. Hyun-Jae Shin, Ph. D. Department of Chemical Engineering Graduate School of Chosun University

Eschericia coli expression vector system with IPTG induction system has been frequently used to produce the human bone morphogenetic protein-2 (hBMP-2) but the inducer price limits the mass production of hBMP-2. In this study, the hBMP-2 was expressed in recombinant E. coli JM83 using a high constitutive expression system (HCE) derived upstream from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and biologically active recombinant hBMP-2 (dimerization yield of rhBMP-2 was 23%) has been produced through conventional in vitro refolding steps. Dimerized rhBMP-2 was purified by heparin-affinity chromatography. The biological effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity assay and by the change of cellular morphology using MC3T3-E1 and ATDC5 cells.

Key words : rhBMP-2; in vitro refolding; constitutive expression system; alkaline phosphatase; osteoblast; morphology