A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex

Honey, S., Schneider, B. L., Schieltz, D. M., Yates, J. R., Futcher, B. (2001) A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex. Nucleic Acids Res, 29 (4). E24. ISSN 1362-4962 (Electronic)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/11160944
DOI: 10.1093/nar/29.4.e24

Abstract

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Item Type: Paper
Uncontrolled Keywords: Adenosine Triphosphatases Amino Acid Sequence Antibodies, Monoclonal/immunology Base Sequence Blotting, Western CDC28 Protein Kinase, S cerevisiae/chemistry/genetics/isolation & purification/ metabolism Calmodulin/metabolism Calmodulin-Binding Proteins/genetics/metabolism Cell Cycle Proteins Chromatography, Affinity/methods Cyclin B/genetics/isolation & purification/ metabolism Fungal Proteins/chemistry/genetics/isolation & purification/metabolism HSP70 Heat-Shock Proteins/chemistry/genetics/isolation & purification/metabolism Hemagglutinins/genetics/immunology Histidine/genetics/metabolism Macromolecular Substances Mass Spectrometry Molecular Sequence Data Molecular Weight Mutagenesis, Insertional Nickel/metabolism Precipitin Tests Protein Binding Recombinant Fusion Proteins/ chemistry/isolation & purification/ metabolism Saccharomyces cerevisiae/genetics/metabolism Saccharomyces cerevisiae Proteins
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
Investigative techniques and equipment > spectroscopy > mass spectrometry
biotechnology > chromatography > protein purification
CSHL Authors:
Communities: CSHL labs > Futcher lab
Depositing User: Matt Covey
Date Deposited: 22 Jan 2014 16:12
Last Modified: 22 Jan 2014 16:12
PMCID: PMC29631
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29254

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