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Nucleic Acids Research, 2001, Vol. 29, No. 4 e24
© 2001 Oxford University Press

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

Sangeet Honey, Brandt L. Schneider, David M. Schieltz1, John R. Yates1 and Bruce Futcher*

Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724, USA and 1Department of Molecular Biotechnology, University of Washington, Health Sciences Center, Seattle, WA 98195, USA

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.

* To whom correspondence should be addressed at present address: Department of Molecular Genetics and Microbiology, SUNY Stony Brook, New York, NY 11794-5222, USA. Tel: +1 631 632 4715; Fax: +1 631 632 9797; Email: bfutcher{at}ms.cc.sunysb.edu Present address Brandt L. Schneider, Department of Cell Biology and Biochemistry, Texas Tech Health Sciences Center, Lubbock, TX 79430, USA


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