Methods and Protocols, Volume 3, Issue 4
December 2020 - 21 articles
Cover Story: E. coli strains and T7-promoter-based plasmids have been widely used for the expression of recombinant proteins. However, the limited amount of soluble protein fused with core-streptavidin made it challenging for the immobilization of protein on biotinylated materials. In this study, a T7-promoter-based pET-30a(+) plasmid encoded with a chimeric thymidine phosphorylase (TP)-coreSA gene was constructed as a model system for the expression of TP-coreSA fusion protein. Various bacterial strains with the T7 expression system were used to analyze the expression of soluble fusion protein, with the goal of minimizing time- and cost-consuming steps of protein purification. Our results indicate that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system could provide efficient expression of soluble TP-coreSA fusion protein for purification. The eluted fusion protein tethered on biotinylated A549 cancer cells revealed its functionality by effectively killing cells after loading with prodrug 5′-DFUR.
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