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.

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.