Purification and crystallization of the CDK-associated protein phosphatase KAP expressed in Escherichia coli.
The kinase associated phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates the cell cycle control protein, cyclin dependent kinase-2 on Thr 160 in a cyclin dependent manner (Poon & Hunter, 1995). We report here the over-expression of KAP in Escherichia coli as an N-terminal His-tagged protein using a modified pET-28a T7-expression vector. The recombinant protein was purified to homogeneity and crystallized. The crystals diffract to 2.3 A resolution when exposed to synchrotron radiation and belong to space group P6(1)22, or its enantiomorph P6(5)22, with unit cell dimensions a = b = 74.5 A, c = 139.5 A.
Hanlon
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Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Barford
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D000595:Amino Acid Sequence; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D051357:Cyclin-Dependent Kinase 2; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004274:DNA, Recombinant; D054637:Dual-Specificity Phosphatases; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C084429:CDKN3 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases