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Title: Cloning and functional characterization of Polo like kinase CaCdc5 from Candida albicans
Researcher: Deshta;Ms Ankita
Guide(s): Sourirajan; Prof. Anuradha
Keywords: CaCdc5
Candida albicans
Ser/Thr kinases
University: Shoolini University of Biotechnology and Management Sciences
Completed Date: 22-06-2016
Abstract: newline ABSTRACT newlineAll the members of Polo-like kinases (PLKs) are characterized by the presence of kinase domain newlinetowards the N- terminus and a conserved polo box domain (PBD) towards the C- terminus, newlineconsisting of two polo boxes. PLKs are one of the master regulators of cell cycle, in addition to newlinecyclin dependent kinase (CDKs). PLKs have been identified in most eukaryotes, except plants. newlineIn lower eukaryotes, such as Candida albicans,a single PLK member named as CaCdc5 exists. newlineInterestingly, depletion of CaCdc5 results in transition of C. albicans from yeast to filamentous newlinegrowth in addition to the cell cycle defects. The present study was undertaken to characterise the newlinePLK CaCdc5 from C. albicans in vitro. newlineFor recombinant expression of CaCdc5, CaCDC5 was amplified by PCR from C. newlinealbicans genomic DNA and ligated to expression vector, pET28a to express CaCdc5 as His6 newlineCaCdc5 fusion protein. However, the recombinant protein, His6-CaCdc5 was not found in the newlineinduced fractions in either of the conditions used for induction, including different temperature newlineand IPTG concentrations. Therefore, CaCDC5 was cloned into pGEX4T2 vector to achieve newlineexpression of CaCdc5 as GST fusion (GST-CaCdc5) and transformed into E. coli strain DH5and#945;. newlineMaximum and soluble expression of GST-CaCdc5 (~ 101 kDa) was obtained in BL21- DE3RIL newlinewhen induced with 1 mM IPTG at 18 and#8304;C for 15 h.GST-CaCdc5 protein was purified from the newlinetotal protein extracts of the induced cells by affinity chromatography with glutathione sepharose newlinefollowed by Fast performance liquid chromatography (FPLC). After purification, GST-CaCdc5 newlinewas analyzed by 10 % SDS-PAGE and western blotting using anti-GST antibodies to confirm newlinethe expression of active protein. After FPLC purification, GST-CaCdc5 was found to be ~ 95 % newlinepure. newlineix newlineGST-CaCdc5 was further analyzed for its functional kinase activity. Since, CaCdc5 is a newlinemember of PLK family, its ability to mediate phosphorylation was tested in vitro using casein as newlinea generic substrate. GST-CaCdc5was found to mediate phosphorylation of casein, indicating that newlinerecombinant CaCdc5 is functionally active. Interestingly, GST-CaCdc5 also exhibited autophosphorylation newlineactivity, a characteristic of many kinases, including PLKs. The kinetics of newlinecasein phosphorylation was linear till ~ 50 minutes, after which saturation was observed, newlineindicating the optimum time of reaction to be ~ 60 minutes. GST-CaCdc5 exhibited an optimum newlinetemperature of 30 and#8304;C and pH 9 for its kinase activity. The phosphorylation of casein was newlinemaximum with 1.3 mM GST-CaCdc5. Titration of the kinase reaction with increasing newlineconcentrations of the substrate casein showed that GST-CaCdc5 exhibits a hyperbolic kinetics, newlineconsistent with Michaelis-Menten behavior. Casein phosphorylation was maximum with 0.35 newlineand#956;M of casein with a Km of 12 nM for casein. On the other hand, addition of increasing newlineconcentrations of unlabeled ATP (0.05 -1.50 mM) did not show significant inhibition of the newlinekinase activity of GST-CaCdc5. Effect of divalent cations like Mg2+, Mn2+ and Ca2+ were newlinestudied. The absence of Mn2+in the kinase reaction drastically reduced the casein newlinephosphorylation activity of GST-CaCdc5, while omission of Mg2+ had no significant effect on newlinekinase activity. The activating effect of Mn2+ was reversed by the addition of metal chelators like newlineEDTA and EGTA. While the addition of 0.1% SDS completely inhibited the kinase activity of newlineGST-CaCdc5, addition of 5 mM DTT did not have significant effect as compared to the standard newlinereaction. Thus, this study showed that recombinant CaCdc5 exhibits Mn2+dependent kinase newlineactivity, which is the first such report on aMn2+dependent PLK.
Pagination: ix,98
Appears in Departments:Faculty Of Biotechnology

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10 review of literature.pdfAttached File414.51 kBAdobe PDFView/Open
11.material and methods.pdf131.55 kBAdobe PDFView/Open
12.results.pdf1.19 MBAdobe PDFView/Open
13.discussion.pdf77.58 kBAdobe PDFView/Open
14.reccomendations.pdf38.25 kBAdobe PDFView/Open
15.summary.pdf76.94 kBAdobe PDFView/Open
16.refrences.pdf178.33 kBAdobe PDFView/Open
17. appendix.pdf211.3 kBAdobe PDFView/Open
18. publication.pdf60.13 kBAdobe PDFView/Open
19.functions of polo-like kinases_ a journey from yeast to humans.pdf3.69 MBAdobe PDFView/Open
1.title.pdf55.73 kBAdobe PDFView/Open
20.paper.pdf576.51 kBAdobe PDFView/Open thesis certificates.pdf45.24 kBAdobe PDFView/Open
3.contents.pdf26.07 kBAdobe PDFView/Open
4.acknowledgement.pdf41.05 kBAdobe PDFView/Open
5.abbreviations.pdf58.46 kBAdobe PDFView/Open
6.list of tables.pdf28.93 kBAdobe PDFView/Open
7.list of figures.pdf50.91 kBAdobe PDFView/Open
8.abstract.pdf59.02 kBAdobe PDFView/Open
9.introduction.pdf331.88 kBAdobe PDFView/Open

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