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- W2179733144 abstract "Cdc25C is a member of the dual specific protein phosphatase family capable of dephosphorylating both phospho-Tyr and phospho-Ser/Thr residues. In vertebrates, there are three isoforms of the Cdc25 enzyme (Cdc25A, Cdc25B and Cdc25C). The N-terminal (regulatory domain) region of these three enzymes is highly divergent (~20-25% identity) while the C-terminal (catalytic domain) region is more conserved (~60% identity). The Cdc25C isoform is of particular importance because it dephosphorylates Cdc2, which is part of the Cdc2/Cyclin B complex, allowing the cell to transition into mitosis. We want to gain further insight into how Cdc25C interacts with its substrate Cdc2. One way to do this is to utilize the ability of E. coli to heterologously express recombinant Cdc25c. This will allow us to obtain large amounts of the protein for in vitro characterization. First, several E. coli cell lines were tested (each with unique properties; e.g. expression of toxic protein, expression of disulfide rich proteins) for their ability to overexpress Cdc25C. The cell line and expression condition having the highest yield of full-length protein was chosen and purified using a single immobilized metal affinity chromatography (IMAC) column that will bind a 6xHis affinity tag fused to the N-terminus of Cdc25C or a tandem approach that utilizes two affinity tags (an N-terminal 6xHis affinity tag and a C-terminal StrepII tag). The pure protein will then be subjected to a variety of chromatography techniques such as UV-Vis spectroscopy and circular dichrosim (CD) to characterize structural changes the protein undergoes when binding to its zinc cofactor. Site directed mutagenesis will be used to create mutations in the zinc binding region of Cdc25C and these proteins will also be characterized for their ability to bind or not to bind the zinc cofactor." @default.
- W2179733144 created "2016-06-24" @default.
- W2179733144 creator A5055037292 @default.
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- W2179733144 date "2014-01-01" @default.
- W2179733144 modified "2023-09-25" @default.
- W2179733144 title "Optimizing The Expression And Purification Of Eukaryotic Cdc25c In E. Coli" @default.
- W2179733144 doi "https://doi.org/10.5339/qfarc.2014.hbpp0065" @default.
- W2179733144 hasPublicationYear "2014" @default.
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