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|Title:||Recombinant expression and biochemical characterization of Saccharomyces cerevisiae Polo like kinase Cdc5|
|Guide(s):||Sourirajan Dr Anuradha|
|University:||Shoolini University of Biotechnology and Management Sciences|
|Abstract:||newline ABSTRACT newlinePolo like kinases (PLKs) belong to serine/threonine kinase subfamily and characterized by the presence of the signature motif called polo-box domain (PBD). PLK members studied so far have emerged as the conserved regulator of cell cycle and cell division in eukaryotes. PLKs play a vital role in cell cycle progression from G1 till the completion of cytokinesis. The budding yeast Saccharomyces cerevisiae possesses a single PLK member called Cdc5. newlineThe present study was undertaken to understand the biochemical features of budding yeast PLK, Cdc5. For recombinant expression of Cdc5, CDC5 was amplified by PCR from S. cerevisiae genomic DNA and ligated to expression vector, pET28a to express Cdc5 as His6 Cdc5 fusion protein. The recombinant construct, pET28a-CDC5 was transformed into E. coli DH5and#945;. Recombinant plasmid DNA was isolated, verified by restriction digestions and DNA sequencing of CDC5 insert. pET28a-CDC5 was retransformed into expression host strains of E. coli (BL21-DE3, BL21-Codon Plus (CP) and Rossetta). The His6-Cdc5 expression was induced by addition of IPTG and analyzed by SDS-PAGE. The inducible expression of His6-Cdc5 of ~82 kDa was confirmed by Western blotting with anti-His antibodies. However, the recombinant protein, His6-Cdc5 was found entirely in insoluble fractions and negligible in soluble fractions in all the conditions used for induction, including different temperature and IPTG concentrations. newlineConsequently, CDC5 was cloned into pGEX4T2 vector to obtain expression of Cdc5 as GST fusion (GST-Cdc5) and transformed into E. coli strain DH5and#945;. CDC5 sequence in recombinant plasmid was verified by sequencing. For expression of GST-Cdc5, the expression host strains of E. coli BL21-DE3, BL21-CP and Rossetta were transformed with pGEX4T2- CDC5 and induced with inducer, IPTG at 37and#8304;C for 4 h. GST-Cdc5 protein was found in the insoluble pellet fraction when analyzed by SDS-PAGE. Therefore, GST-Cdc5 expression was standardized by alteration of induction conditions (temperature and IPTG concentrations). Maximum and soluble expression of GST-Cdc5 was obtained in BL21-Codon Plus (CP) when induced with 0.1 mM IPTG at 18and#8304;C for 15 h. The expression and size of GST-Cdc5 (~107 kDa) was confirmed by Western analysis with anti-GST antibodies. To achieve purification of recombinant GST-Cdc5 protein, its expression was induced in large scale culture of E. coli BL21-CP newlineviii newlineharboring pGEX4T2-CDC5. GST-Cdc5 protein was purified from the total protein extract of the induced cells by affinity chromatography with glutathione sepharose. After purification, GST-Cdc5 was analyzed by 10 % SDS-PAGE and by Western blotting with anti-GST antibodies to confirm the intactness and purity of the protein. The GST-Cdc5 preparation was ~ 95 % homogeneous. newlineThe purified GST-Cdc5 was analyzed for functional activity. Since Cdc5 is a kinase, it must exhibit phosphorylation of appropriate substrates. For functional validation of purified GST-Cdc5, in vitro casein phosphorylation assay was performed. GST-Cdc5 was found to be functionally active. It mediated phosphorylation of casein in vitro. Interestingly, GST-Cdc5 exhibited substrate (casein) as well as auto-phosphorylation, a characteristic of PLKs. After the functional validation of GST-Cdc5, the biochemical features of in vitro kinase activity of GST- Cdc5 were characterized. Quantification of the intensity of casein phosphorylation showed that the kinetics of phosphorylation was linear till ~ 45 minutes, after which saturation was observed, indicating the optimum time of reaction to be ~ 45 minutes. The casein phosphorylation by GST- Cdc5 was optimal at temperature of 30and#8304;C and pH of 9.0. The phosphorylation was maximum with 2.7 and#956;M GST-Cdc5. GST-Cdc5 exhibited a Km of 1.35 and#956;M for casein. While Mg2+ (at two times concentration as compared to concentration in the standard reaction) served as an activator of kinase activity of GST-Cdc5, Mn2+ (at two times concentration) and Ca2+ had no effect on the kinase activity. In addition to casein, GST-Cdc5 exhibited phosphorylation on other substrates like BSA and Myelin basic protein. newlineIn future, the recombinant GST-Cdc5 can be used as an amenable tool for in vitro pull down of its substrates from yeast, mutational analysis for structural and functional analysis, and consensus sites determination of its substrates, which are still at infancy. These findings can be directly extended to PLKs in humans because of close structural and functional conservation of PLKs. In humans Plk1 is implicated in tumorigenesis, so currently it is being pursued extensively for the development of antiproliferative drugs.|
|Appears in Departments:||Faculty Of Biotechnology|
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