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A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to ot...

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Detalles Bibliográficos
Autores principales: Hayashi, Hiroki, Kishi, Tsutomu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8707574/
https://www.ncbi.nlm.nih.gov/pubmed/34946108
http://dx.doi.org/10.3390/microorganisms9122505
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author Hayashi, Hiroki
Kishi, Tsutomu
author_facet Hayashi, Hiroki
Kishi, Tsutomu
author_sort Hayashi, Hiroki
collection PubMed
description Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.
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spelling pubmed-87075742021-12-25 A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae Hayashi, Hiroki Kishi, Tsutomu Microorganisms Article Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins. MDPI 2021-12-03 /pmc/articles/PMC8707574/ /pubmed/34946108 http://dx.doi.org/10.3390/microorganisms9122505 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hayashi, Hiroki
Kishi, Tsutomu
A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title_full A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title_fullStr A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title_full_unstemmed A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title_short A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae
title_sort set of plasmid-based modules for easy switching of c-terminal epitope tags in saccharomyces cerevisiae
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8707574/
https://www.ncbi.nlm.nih.gov/pubmed/34946108
http://dx.doi.org/10.3390/microorganisms9122505
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