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A vector system for efficient and economical switching of C‐terminal epitope tags in Saccharomyces cerevisiae
Author(s) -
Sung MinKyung,
Ha Cheol Woong,
Huh WonKi
Publication year - 2008
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1588
Subject(s) - epitope , biology , ura3 , plasmid , saccharomyces cerevisiae , fusion protein , kluyveromyces lactis , gene , yeast , computational biology , oligonucleotide , genetics , microbiology and biotechnology , recombinant dna , antigen
In Saccharomyces cerevisiae , one‐step PCR‐mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C‐ or N‐terminus. For many purposes, C‐terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C‐terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module‐based C‐terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C‐terminal epitope tagging of proteins in yeast cells that already carry MX6 module‐based gene deletion or C‐terminal epitope tag. Copyright © 2008 John Wiley & Sons, Ltd.