Chinese Journal of Biotechnology 2002;18(3):339-342

Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli.

Tao CHEN 1 ; Rui-Song YU ; Hui-Li LIU ; Zhen LI ; Xiang-Rong CAO

Affiliations

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Country

China

Language

Chinese

Abstract

By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E. coli. The expression plasmid pGEX-IFN was constructed successfully. Recombinant porcine IFN alpha, which is expressed as inclusion bodies, was about 20% of the total proteins. The inclusion body was dissolved in 8 mol/L urea and subsequently renatured by dilution in refolding buffer. In order to obtain pure protein, the renatured IFN alpha was purified by FPLC, and the cytokine activity (5200 IU/mg) was verified by inhibiting the cytopathic effect.