ISSN:
0749-503X
Keywords:
Schizosaccharomyces pombe
;
cell cycle
;
Cdc2 kinase
;
GST
;
Life and Medical Sciences
;
Genetics
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
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.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/yea.320101212
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