Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe

Carmel, G., Leichus, B., Cheng, X., Patterson, S. D., Mirza, U., Chait, B. T., Kuret, J. (1994) Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe. J Biol Chem, 269 (10). pp. 7304-9. ISSN 0021-9258 (Print)

URL: http://www.ncbi.nlm.nih.gov/pubmed/8125945

Abstract

The catalytic domain of Schizosaccharomyces pombe casein kinase-1 (the product of the cki1 gene) has been overexpressed in Escherichia coli, purified by chromatographic methods, characterized in vitro, and crystallized in the presence and absence of nucleotide substrate. The best crystals belong to the trigonal space group P3(1)21 or its enantiomorph, have unit cell parameters a = b = 79 A, c = 121 A, and diffract x-rays to 2.0-A resolution. Kinetic characterization of the purified catalytic domain and other C-terminal deletion mutants of Cki1 suggests that it is subject to two forms of regulation. One mechanism involves autophosphorylation, and results in a 4-fold decrease in the affinity for protein substrate. In contrast, truncation of intact Cki1 results in a 3-fold activation in its catalytic rate. This activation may arise from the removal of an inhibitory domain present in the intact enzyme.

Item Type: Paper
Uncontrolled Keywords: Amino Acid Sequence Casein Kinases Catalysis Chromatography, Ion Exchange Cloning, Molecular Crystallography, X-Ray Escherichia coli/genetics Kinetics Molecular Sequence Data Mutation Protein Conformation Protein Kinases/chemistry/ genetics/isolation & purification Research Support, U.S. Gov't, P.H.S. Schizosaccharomyces/ enzymology Sequence Deletion Spectrum Analysis, Mass
Subjects: Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase
Investigative techniques and equipment > spectroscopy > mass spectrometry
Investigative techniques and equipment > x ray crystallography
organism description > yeast
CSHL Authors:
Communities: CSHL labs > Stillman lab
Depositing User: Matt Covey
Date Deposited: 07 May 2015 19:47
Last Modified: 07 May 2015 19:47
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31457

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