Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli.
- Author:
Tao CHEN
1
;
Rui-Song YU
;
Hui-Li LIU
;
Zhen LI
;
Xiang-Rong CAO
Author Information
1. School of Life Science, Nanjing Normal University, Nanjing 210097, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
Interferon-alpha;
biosynthesis;
isolation & purification;
Mutagenesis, Site-Directed;
Protein Precursors;
biosynthesis;
isolation & purification;
Recombinant Proteins;
biosynthesis;
isolation & purification
- From:
Chinese Journal of Biotechnology
2002;18(3):339-342
- CountryChina
- 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.
