for profiling the yeast proteome
To facilitate global protein analyses, Dr. Erin O'Shea and Dr. Jonathan
Weissman (UCSF) have created a library where each ORF is tagged with a
high-affinity epitope and expressed from its natural chromosomal
location. Through immunodetection of the common tag, a resource now
exists that provides a census of proteins expressed during log-phase
growth and quantifies their absolute levels. Characterization of this
library revealed that ~80% of the proteome is expressed during normal
growth conditions. The abundance of proteins ranged from fewer than 50
to more than 106 molecules/cell with many, including essential proteins
and most transcription factors, having levels not readily detectable by
other proteomic techniques, nor predictable by mRNA levels or codon
bias measurements (Figures 1 & 2)1.
The Yeast-TAP-Fusion Library allows the purification and selection of
the entire yeast proteome and associated components using two simple
affinity selection steps in tandem, enabling the development of a range
of high-throughput functional assays. A collection of ORF specific
oligonucleotide primers were synthesized. Each primer pair possesses
shared 3' ends that allow for PCR amplification of a common insertion
cassette, as well as gene-specific 5' ends that allow for the precise
introduction, through homologous recombination, of the amplified
insertion cassettes as a perfect in-frame fusion at the C-terminal end
of the coding region of each gene (Figure 3).
The C-terminal TAP insertion cassette contains the coding region
for a modified version of the TAP (Tandem Affinity Purification) tag,
which consists of a calmodulin binding peptide, a TEV cleavage site and
two IgG binding domains of Staphylococcus aureus protein A, as well as
a selectable marker. Background strain = MatA (BY4741). The Anti-TAP antibody is highly specific for the TAP.
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Shipping Information:
Individual strains are provided in tubes containing glycerol stock
cultures in YPD plus 15% glycerol. While the scTAP strains are supplied
in YPD media, SD complete or SD -HIS media can also be used. Individual
strains from this library can be queried and purchased through our
clone query.
The TAP Library is provided in 96-well microtiter plates containing
frozen glycerol stock cultures in YPD plus 15% glycerol. While the
scTAP strains are supplied in YPD media, SD complete or SD -HIS media
can also be used. Strains are arranged in plates by protein expression
size from largest to smallest, with plate designation GS1 representing
high expression and GS5 representing low expression. In addition a
membrane bound protein subset has been arrayed by Open Biosystems in
separate plates for your convenience. Refer to CD shipped with the
collection for expression category designation.
References:
1Ghaemmaghami, S., Huh, W., Bower, K., Howson, R., Belle, A.,
Dephoure, N., O'Shea, E. and Weissman, J. Global Analysis of Protein
Expression in Yeast. Nature, 425, 737 - 741 (16 Oct 2003) Letters to
Nature .
The use of the TAP-tag for the purification of biomolecule /protein
complexes is covered by granted patents in Europe and Australia
(EP1105508B1, AU07629621) and patent applications in Canada, Japan and
US (see int. pat. appl . WO-0009716).
For licensing information for use in purification of biomolecule
/protein complexes, please contact Cellzome AG, Meyerhofstr . 1, 69117
Heidelberg , Germany at info@cellzome.com.
For commercial licensing information regarding further fields of use,
please contact Technology Officer, Open Biosystems, Inc., 601 Genome Way, Suite 2100, Huntsville , AL 35806 . Phone: 256-704-4848 Fax
: 256-704-4849 .
