Article

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

School of Life Science, Nanjing Normal University, Nanjing 210097, China.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2002; 18(3):339-42. pp.339-42
Source: PubMed

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.

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Keywords

8 mol/L urea
 
bias code
 
cytokine activity
 
expression plasmid pGEX-IFN
 
huge primer PCR Cys86
 
PoIFN-alpha
 
Recombinant porcine IFN alpha
 
refolding buffer
 
renatured
 
renatured IFN alpha
 
total proteins
 
Tyr(TAC)