[Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli].
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.
Chen
Tao
T
School of Life Science, Nanjing Normal University, Nanjing 210097, China.
Yu
Rui-Song
RS
Liu
Hui-Li
HL
Li
Zhen
Z
Cao
Xiang-Rong
XR
D004926:Escherichia coli / Q000235:genetics*; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D016898:Interferon-alpha; D011498:Protein Precursors; D011994:Recombinant Proteins