Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: a new approach to the study of cell cycle control proteins.
Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.
Leroy
D
D
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Toulouse, France.
Baldin
V
V
Ducommun
B
B
D016203:CDC2 Protein Kinase / Q000096:biosynthesis* / Q000302:isolation & purification; D002453:Cell Cycle; D019926:Cyclin B; D016213:Cyclins / Q000032:analysis / Q000494:pharmacology; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000235:genetics*
D019926:Cyclin B; D016213:Cyclins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase