Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.
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.
Howson
Russell
R
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California at San Francisco, 600 16th Street, Genentech Hall, San Francisco, CA 94143-2240, USA.
Huh
Won-Ki
WK
Ghaemmaghami
Sina
S
Falvo
James V
JV
Bower
Kiowa
K
Belle
Archana
A
Dephoure
Noah
N
Wykoff
Dennis D
DD
Weissman
Jonathan S
JS
O'Shea
Erin K
EK