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. 2005 Feb-Mar;6(1-2):2–16. doi: 10.1002/cfg.449

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

Russell Howson 1, Won-Ki Huh 1,3, Sina Ghaemmaghami 2, James V Falvo 1, Kiowa Bower 2, Archana Belle 1, Noah Dephoure 1, Dennis D Wykoff 1, Jonathan S Weissman 2, Erin K O'Shea 1,✉
PMCID: PMC2448600  PMID: 18629296

Abstract

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebersold Ruedi, Mann Matthias. Mass spectrometry-based proteomics. Nature. 2003 Mar 13;422(6928):198–207. doi: 10.1038/nature01511. [DOI] [PubMed] [Google Scholar]
  2. Brachmann C. B., Davies A., Cost G. J., Caputo E., Li J., Hieter P., Boeke J. D. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998 Jan 30;14(2):115–132. doi: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  3. Cliften Paul, Sudarsanam Priya, Desikan Ashwin, Fulton Lucinda, Fulton Bob, Majors John, Waterston Robert, Cohen Barak A., Johnston Mark. Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science. 2003 May 29;301(5629):71–76. doi: 10.1126/science.1084337. [DOI] [PubMed] [Google Scholar]
  4. DeRisi J. L., Iyer V. R., Brown P. O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science. 1997 Oct 24;278(5338):680–686. doi: 10.1126/science.278.5338.680. [DOI] [PubMed] [Google Scholar]
  5. Diehn M., Eisen M. B., Botstein D., Brown P. O. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nat Genet. 2000 May;25(1):58–62. doi: 10.1038/75603. [DOI] [PubMed] [Google Scholar]
  6. Ghaemmaghami Sina, Huh Won-Ki, Bower Kiowa, Howson Russell W., Belle Archana, Dephoure Noah, O'Shea Erin K., Weissman Jonathan S. Global analysis of protein expression in yeast. Nature. 2003 Oct 16;425(6959):737–741. doi: 10.1038/nature02046. [DOI] [PubMed] [Google Scholar]
  7. Haswell E. S., O'Shea E. K. An in vitro system recapitulates chromatin remodeling at the PHO5 promoter. Mol Cell Biol. 1999 Apr;19(4):2817–2827. doi: 10.1128/mcb.19.4.2817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huh Won-Ki, Falvo James V., Gerke Luke C., Carroll Adam S., Howson Russell W., Weissman Jonathan S., O'Shea Erin K. Global analysis of protein localization in budding yeast. Nature. 2003 Oct 16;425(6959):686–691. doi: 10.1038/nature02026. [DOI] [PubMed] [Google Scholar]
  9. Kellis Manolis, Patterson Nick, Endrizzi Matthew, Birren Bruce, Lander Eric S. Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature. 2003 May 15;423(6937):241–254. doi: 10.1038/nature01644. [DOI] [PubMed] [Google Scholar]
  10. Lockhart D. J., Dong H., Byrne M. C., Follettie M. T., Gallo M. V., Chee M. S., Mittmann M., Wang C., Kobayashi M., Horton H. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol. 1996 Dec;14(13):1675–1680. doi: 10.1038/nbt1296-1675. [DOI] [PubMed] [Google Scholar]
  11. Longtine M. S., McKenzie A., 3rd, Demarini D. J., Shah N. G., Wach A., Brachat A., Philippsen P., Pringle J. R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998 Jul;14(10):953–961. doi: 10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  12. MacBeath G., Schreiber S. L. Printing proteins as microarrays for high-throughput function determination. Science. 2000 Sep 8;289(5485):1760–1763. doi: 10.1126/science.289.5485.1760. [DOI] [PubMed] [Google Scholar]
  13. Martzen M. R., McCraith S. M., Spinelli S. L., Torres F. M., Fields S., Grayhack E. J., Phizicky E. M. A biochemical genomics approach for identifying genes by the activity of their products. Science. 1999 Nov 5;286(5442):1153–1155. doi: 10.1126/science.286.5442.1153. [DOI] [PubMed] [Google Scholar]
  14. Phizicky Eric, Bastiaens Philippe I. H., Zhu Heng, Snyder Michael, Fields Stanley. Protein analysis on a proteomic scale. Nature. 2003 Mar 13;422(6928):208–215. doi: 10.1038/nature01512. [DOI] [PubMed] [Google Scholar]
  15. Schena M., Shalon D., Davis R. W., Brown P. O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 1995 Oct 20;270(5235):467–470. doi: 10.1126/science.270.5235.467. [DOI] [PubMed] [Google Scholar]
  16. Tong A. H., Evangelista M., Parsons A. B., Xu H., Bader G. D., Pagé N., Robinson M., Raghibizadeh S., Hogue C. W., Bussey H. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 2001 Dec 14;294(5550):2364–2368. doi: 10.1126/science.1065810. [DOI] [PubMed] [Google Scholar]
  17. Zhu H., Bilgin M., Bangham R., Hall D., Casamayor A., Bertone P., Lan N., Jansen R., Bidlingmaier S., Houfek T. Global analysis of protein activities using proteome chips. Science. 2001 Jul 26;293(5537):2101–2105. doi: 10.1126/science.1062191. [DOI] [PubMed] [Google Scholar]

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