Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

Yeast
Joseph J TastoKathleen L Gould

Abstract

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.

References

Jan 1, 1991·Methods in Enzymology·S MorenoP Nurse
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Citations

Feb 21, 2007·Nature Structural & Molecular Biology·Shane M BukerDanesh Moazed
Mar 9, 2004·Molecular Biology of the Cell·Srinivas VenkatramKathleen L Gould
Sep 24, 2004·Molecular Biology of the Cell·Hanbing AnKathleen L Gould
Jul 20, 2012·Molecular Biology of the Cell·Yamei WangQuan-wen Jin
Aug 11, 2007·Nucleic Acids Research·Naomi FershtPaul Nurse
Feb 20, 2007·Molecular and Cellular Biology·Cosmin SaveanuMicheline Fromont-Racine
Jul 30, 2004·Molecular and Cellular Biology·David W PowellAndrew J Link
Dec 31, 2005·Molecular and Cellular Biology·Nina B LeedsJonathan P Staley
Dec 7, 2011·PLoS Genetics·Caia D S Duncan, Juan Mata
Apr 9, 2008·Proceedings of the National Academy of Sciences of the United States of America·Monique DewezDamien Hermand
May 10, 2003·Proceedings of the National Academy of Sciences of the United States of America·Camilla O SamuelsenClaes M Gustafsson
Jun 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Michael J MacCossJohn R Yates
Mar 16, 2007·Proceedings of the National Academy of Sciences of the United States of America·Melanie D OhiThomas Walz
Mar 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·Jaya K KumarCharles C Richardson
Apr 24, 2014·BMC Genomics·Caia D S Duncan, Juan Mata
Jun 9, 2004·The Journal of Cell Biology·Becket FeierbachFred Chang
Jan 14, 2009·The Journal of Cell Biology·Rachel H Roberts-GalbraithKathleen L Gould
Nov 30, 2006·The Journal of Cell Biology·Marcus B SmolkaHuilin Zhou

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