Type II proteinase inhibitors (PI2) derived from potato tubers have demonstrated protective capabilities against microbes and insects through the inhibition of foreign proteases. This family of protease inhibitors also display anticancer activity and hold potential therapeutic applications in multiple cancer models. We report here the cloning of a genetically-engineered PI2 gene variant, pi2E.coli*c.994_1110del encoding Pi2K, into different expression vectors encoding one of two affinity tags, a mannose-binding protein (MBP) or a hexahistidine tag (His-tag). Growth of Escherichia coli strains DH5α and BL21(DE3), transformed with the constructed plasmids, suggest that tagged Pi2K is not toxic. Purification using amylose resin and SDS-PAGE analysis of recovered fractions revealed that the MBPPi2K fusion protein is soluble and may be susceptible to degradation. These results provide a framework to explore the biochemical inhibitory activity of Pi2K to determine its specific activity and substrate range. Together, our data point to the capacity of E. coli BL21(DE3) strain for heterologous expression of soluble Pi2K and further studies of the biotechnological and therapeutic value of this proteinase inhibitor.
Expression and Purification of a Potato Type II Proteinase Inhibitor in Escherichia coli Strain BL21(DE3)
Fall 2015 / Winter 2016