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

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

doi:10.1371/journal.pone.0176184

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.

Highlights

The budding yeast Saccharomyces cerevisiae has been widely used in basic research due to the ease with which it can be genetically manipulated
To solve the two main problems of an inability to recycle the selectable marker and the presence of “scar” sequences after tagging, we proposed that the URA3 flanking sequences hisG in the traditional hisG-URA3-hisG system [9] could be replaced by the desired genetic element in tandem repeats
We chose three typical fluorescent protein (FP): mCherry, sfGPF, and mKikGR, as tags. mCherry is a red monomer that is widely used due to it having the best photostability among the red FPs [23]. sfGFP is an engineered green fluorescent protein (GFP) with increased resistance to denaturation and improved folding kinetics [24]

Summary

Introduction

The budding yeast Saccharomyces cerevisiae has been widely used in basic research due to the ease with which it can be genetically manipulated. Considerable progress has been made in studies on budding yeast via the use of epitope and fluorescent protein (FP) tags. Epitope tags, such as Flag, HA, and Myc, are useful for western blotting analysis, immunoprecipitation, affinity purification, and immunochemistry [1,2,3]. FP tags, such as green fluorescent protein (GFP) and its variants, are extensively used in molecular and cellular biology studies. The global protein subcellular localization was studied by plasmid-based epitope-tagging and immunofluorescence analyses [4], or by fusing a GFP tag at the C-terminal of the coding region of a gene of interest, followed by microscopic imaging [5]

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Conclusion