A method for labeling proteins with tags at the native genomic loci in budding yeast

Qian Wang; Huijun Xue; Siqi Li; Ying Chen; Xuelei Tian; Xin Xu; Wei Xiao; Yu Vincent Fu

doi:10.1371/journal.pone.0176184

A method for labeling proteins with tags at the native genomic loci in budding yeast

PLoS One. 2017 May 1;12(5):e0176184. doi: 10.1371/journal.pone.0176184. eCollection 2017.

Authors

Qian Wang¹, Huijun Xue^{2

3}, Siqi Li¹, Ying Chen¹, Xuelei Tian^{1

4}, Xin Xu², Wei Xiao^{1

5}, Yu Vincent Fu^{2

3}

Affiliations

¹ College of Life Sciences, Capital Normal University, Beijing, China.
² State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
³ Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China.
⁴ National Institute for Radiological Protection, China CDC, Beijing, China.
⁵ Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK Canada.

Abstract

Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a "scar" sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.

MeSH terms

Blotting, Western
Epitopes / genetics
Epitopes / metabolism
Genetic Loci*
Genetic Techniques*
Genome, Fungal*
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Microscopy, Fluorescence
Protein Domains
Saccharomyces cerevisiae / cytology
Saccharomyces cerevisiae / genetics*
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / genetics*
Saccharomyces cerevisiae Proteins / metabolism

Substances

Epitopes
Luminescent Proteins
Saccharomyces cerevisiae Proteins

Grants and funding

This study was supported by the National Natural Science Foundation of China grant 31371264 for (YVF), the National Natural Science Foundation of China, 31670068 for (XW), the the Newton Advanced Fellowship (NA140085) for (YVF) from the Royal Society and CAS Interdisciplinary Innovation Team for (YVF). It was partially funded by Student's Platform for Innovation and Entrepreneurship Training Program for (QW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.