Journal Article

A set of epitope-tagging integration vectors for functional analysis in <i>Saccharomyces cerevisiae</i>

Hyeran Sung, Kyung Chul Han, Jun Chul Kim, Ki Wan Oh, Hwan Su Yoo, Jin Tae Hong, Youn Bok Chung, Chong-kil Lee, Kyung S. Lee and Sukgil Song

in FEMS Yeast Research

Volume 5, issue 10, pages 943-950
Published in print July 2005 |
Published online April 2006 | e-ISSN: 1567-1364 | DOI: http://dx.doi.org/10.1016/j.femsyr.2005.03.008

Show Summary Details

Preview

Abstract

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.

Keywords: Saccharomyces cerevisiae; Epitope tagging; Integration vector

Journal Article.  3980 words.  Illustrated.

Full text: subscription required

How to subscribe Recommend to my Librarian

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.