title: A novel approach to the expression and purification of recombinant Xenopus Cdc25C.
abstract: The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family.
authors.affiliation: Weill Cornell Medicine Qatar, Department of Physiology and Biophysics, Doha, Qatar.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schaefer-Ramadan
authors.affiliation: Weill Cornell Medicine Qatar, Pre-medical Education, Doha, Qatar.
authors.forename: Ghizlaine
authors.initials: G
authors.lastname: Bendriss
authors.affiliation: Weill Cornell Medicine Qatar, Department of Physiology and Biophysics, Doha, Qatar.
authors.forename: Satanay
authors.initials: S
authors.lastname: Hubrack
authors.affiliation: Weill Cornell Medicine Qatar, Pre-medical Education, Doha, Qatar.
authors.forename: Abdulrahman
authors.initials: A
authors.lastname: Al-Abdulmalek
authors.affiliation: Weill Cornell Medicine Qatar, Department of Physiology and Biophysics, Doha, Qatar. Electronic address: Khm2002@qatar-med.cornell.edu.
authors.forename: Khaled
authors.initials: K
authors.lastname: Machaca
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; C583458:Trp-Ser- His-Pro-Gln-Phe-Glu-Lys; D029867:Xenopus Proteins; C000611743:Cdc25C protein, Xenopus; D020687:cdc25 Phosphatases
keywords: Cdc25C; Cell cycle; E. coli optimized; Oocyte; Phosphatase; Xenopus