Purification of Mitotic Phosphatase Cdc14p

Abstract

Two methods were employed to achieve soluble expression of Cdc14p. The first being the retrieval of CDC14 from a pET28 expression plasmid and insertion into a GST plasmid by use of the restriction enzyme NdeI. Before this could occur, alteration of an internal NdeI recognition sequence was attempted via site-directed mutagenesis. The second method being a process of exposing cells containing pET28 to a solution 10% sarkosyl or of 2% sarkosyl 2% triton X-100 and 20 mM CHAPS to disrupt any possible inclusion bodies of properly folded Cdc14p. Once soluble protein is extracted by either method, two assays will be preformed to ensure enzymatic activity and specificity will be examined.