Expression， Purification， and Refolding of Recombinant Human EGF-IL-18 Fusion Protein in E. coli

Abstract: EGF-IL-18 fusion gene was amplified from pFUS-EGF-IL18 by PCR， and then was inserted into pET32a(+) to construct a prokaryotic expression vector pET32a(+)-EGF-IL-18. The target protein was highly expressed in E. coli Rosetta (DE3) and was mainly present in inclusion body observed by SDS-PAGE. After EGF-IL-18 inclusion body was repeatedly washed by 2 mol/L urea and 1%Triton X-100， the denatured EGF-IL-18 was refolded by ion-exchange chromatography using the urea linear gradient strategy. It showed that ion-exchange chromatography not only facilitated the refol ding of EGF-IL-18， but also the target protein was purified at the same time. After being further purified with size-exclusion chromatography， EGF-IL-18 could effectively induce expression of interferon-g mRNA in PBMC in the presence of 0.5 mg/ml ConA in vitro. This simple， effective method may be applied in the future to mass production of EGF-IL-18 and lay a solid base for detailed evaluation of bioactivities of EGF-IL-18 in vitro and in vivo.
    

CSTR: 32200.14.cjcb.2006.05.0015