Exploring the differences between the three pyruvate kinase isozymes from Vibrio cholerae in a heterologous...

The genome of Vibrio cholerae has three paralog genes encoding for distinct pyruvate kinases. We were interested in elucidating whether they were expressed, and contributed to the pyruvate kinase activity of V. cholerae. VcIPK and VcIIPK were transformed and expressed in BL21-CodonPlus(DE3)-RIL strain, whereas VcIIIPK could not be transformed. Those studied did contribute to the pyruvate kinase activity of the bacteria. Therefore, our aim was to find an efficient transformation and commonly used over-expression heterologous system for VcIIIPK and develop its purification protocol.

vcIpk, vcIIpk and vcIIIpk genes were transformed in six different BL21 expression strains. No transformants were obtained for the vcIIIpk gene using BL21(DE3), BL21(DE3)pLysS and BL21(DE3)CodonPlus-RIL strains. Reduced rates of cell growth were observed for BL21-Gold(DE3)pLysS and Origami B(DE3)pLysS. High efficiency of transformation was obtained for BL21-AI. Using this strain, VcIIIPK was purified but proved to be unstable during its purification and storage. Therefore, the transformation of vcIIIpk gene resulted in a toxic, mildly toxic or nontoxic product for these BL21 strains. Despite VcIIPK and VcIIIPK being phylogenetically related, the preservation of the proteins is drastically different; whereas one is preserved during purification and storage, the other is auto-proteolyzed completely in less than a week.