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Volume: 3, Issue: 4, July-August, 2015
DOI: 10.7324/JABB.2015.3405

Research Article

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

Abstract

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 study 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.

References

1. Archambault V, Glover DM. Polo-like kinases: Conservation and divergence in their functions and regulation. Nature Reviews of Molecular Cell Biology. 2009; 10: 265-275.

2. Zitouni S, Nabais C, Jana SC, Guerrero A, Bettencourt-Dias M. Polo-like kinases: structural variations lead to multiple functions. Nature Reviews of Molecular Cell Biology. 2014; 15: 433-452.

3. Nigg EA. Polo-like kinases: positive regulators of cell division from start to finish. Current Opinion of Cell Biology. 1998; 10: 776-783.

4. Fenton B, Glover DM. A conserved mitotic kinase active at late anaphase\—telophase in syncytial Drosophila embryos. Nature. 1993; 363: 637-640.

5. Kitada K, Johnson AL, Johnston LH, Sugino A. A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5, Molecular and Cellular Biology. 1993; 13: 4445-4457.

6. Ohkura H, Hagan IM, Glover DM. The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes and Developement. 1995; 9: 1059-1073.

7. Sakchaisri K, Asano S, Yu LR, Shulewitz MJ, Park CJ, Park JE, Cho YW, Veenstra TD, Thorner J, Lee KS. Coupling morphogenesis to mitotic entry. Proccedings of the National Academy of Sciences of the United State of America. 2004; 101: 4124-4129.

8. Alexandru G, Uhlmann F, Mechtler K., Poupart MA , Nasmyth K. Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast. Cell. 2001; 105:459-472.

9. Lee KS, Park JE, Asano S, Park CJ. Yeast polo-like kinases: functionally conserved multitask mitotic regulators. Oncogene. 2005; 24: 217-29.

10. Lee BH, Amon A. Polo kinase\—meiotic cell cycle coordinator. Cell Cycle. 2003; 2: 400\—402.

11. Sourirajan A, Lichten M. Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis. Genes and Development. 2008; 22: 2627-32.

12. Eckerdt F, Yuan J, Strebhardt K. Polo-like kinases and oncogenesis. Oncogene. 2005; 24: 267-276.

13. Archambault V, Lepine G, Kachaner D. Understanding the polo kinase machine. Oncogene. 2015; 1-9.

14. Mortensen EM, Haas W, Gygi M, Gygi SP, Kellogg DR. Cdc28-Dependent Regulation of the Cdc5/Polo Kinase. Current Biology. 2005; 15:2033-2037.

15. Smith DB, Johnson KS, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988; 67: 31-40.

How to cite this article:
Sujata Chauhan, Subhasis Samanta, Jitendra K Thakur, and Anuradha Sourirajan. Cloning, expression and purification of functionally active Saccharomyces cerevisiae Polo-like Kinase, Cdc5 in E. coli. J App Biol Biotech. 2015; 3 (04): 020-024.