Methods for preparation of proteins and protein complexes that regulate the eukaryotic cell cycle for structural studies.
The determination of structures for proteins that control the eukaryotic cell cycle by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography has made a significant contribution to our understanding of the molecular mechanisms that control cell cycle progression. CDK2 has proved particularly tractable to structural analysis, and CDK2 in complex with various regulatory proteins and in different phosphorylation states provides a paradigm for the control of this important kinase family. This chapter describes a number of protocols that can be used to prepare CDKs and selected CDK binding proteins suitable for structural studies by heterologous expression in either E. coli or insect cells.
Welburn
Julie
J
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK.
Endicott
Jane
J
D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D002460:Cell Line; D018360:Crystallography, X-Ray; D019925:Cyclin A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D006801:Humans; D008958:Models, Molecular; D046912:Multiprotein Complexes; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D018797:Cell Cycle Proteins; D019925:Cyclin A; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C098333:cyclin A3 protein, Bos taurus; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases