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Volume: 3, Issue: 4, July-August, 2015
Cloning, expression and purification of functionally active Saccharomyces cerevisiae Polo-like Kinase, Cdc5 in E. coli
Sujata Chauhan, Subhasis Samanta, Jitendra K Thakur, Anuradha Sourirajan
Polo-like Kinases (PLKs) belong to the serine/threonine family of protein kinases. They govern cell cycle progression in concert with CDKs and other cell cycle kinases in most eukaryotes studied. Polo-box domain, the signature motif of PLKs, functions to recruit the kinase to its substrates. However, the substrate specificity, and the complete repertoire of its substrates is not fully known, and their mode of action is still under investigation. The present astudy was undertaken to clone and express the budding yeast PLK, Cdc5 in E. coli. The recombinant Cdc5 was successfully expressed as a GST-Cdc5 fusion protein of ~ 107 kDa. GST-Cdc5 was purified to ~ 95% homogeneity. Interestingly, the recombinant GST-Cdc5 exhibited kinase activity in vitro. GST-Cdc5, but not GST-tag alone phosphorylated the generic substrate casein as well as showed autophosphorylation of the kinase itself. Thus, the recombinant GST-Cdc5 protein is functionally active in vitro and mimics its characteristics in vivo. The availability of the recombinant and active Cdc5 protein would facilitate structure–function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively for further insight into the cell cycle regulation mechanisms of PLKs.
Keywords: Polo-like kinase, recombinant Cdc5, Serine/threonine kinase, Casein phosphorylation, in vitro kinase activity.