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Comparison of Expression and Activity of Antiviral Protein RC28 in Escherichia coli and Pichia pastoris

HU Li-qiang;ZHENG Wen;ZHONG Yi;DU Dan;YANG Hao;GONG Meng;Hua Xi-Washington Mitochondria and Metabolism Research Center,West China Hospital,SCU;  
RC28 is a novel antiviral protein extracted from the mushroom Rozites caperata. In our previous studies,RC28 c DNA were obtained from Rozites caperata total RNA by 3'-RACE and inserted into p MD18-T plasmid by TA cloning. RC28 c DNA fragments were sub-cloned into E. coli expression vector p ET28a( +) and Pichia yeast expression vector p PIC9 K separately to form recombinant RC28 expression vectors with N'-terminal histidine tags. The two kinds of RC28 expression vectors were transformed into their respective host strains and positive clones were selected to construct stable expression systems. SDA-PAGE and Western blot were performed to analyze RC28 expression after induction. RC28 protein were purified and collected by using Ni-NTA columns and detection of the antiviral activity of the recombinant protein achieved by MTT assay. In our experiment,soluble recombined RC28 proteins were prepared both in E. coli and Pichica yeast and the yields of purified protein from these two expression systems were about 3. 5mg / L and 0. 2mg / L,respectively. However,the recombined RC28 protein expressed from E. coli system showed poor antiviral activity while the recombined RC28 protein from Pichica yeast system showed much higher antiviral activity which was close to that of the natural protein.
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