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A method for labeling proteins with tags at the native genomic loci in budding yeast
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A method for labeling proteins with tags at the native genomic loci in budding yeast
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A method for labeling proteins with tags at the native genomic loci in budding yeast
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Journal Article
A method for labeling proteins with tags at the native genomic loci in budding yeast
2017
Overview
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.
Publisher
Public Library of Science,Public Library of Science (PLoS)
Subject
/ Epitopes
/ Genes
/ Genomes
/ Growth
/ Homology
/ Luminescent Proteins - genetics
/ Luminescent Proteins - metabolism
/ Marking
/ Methods
/ Proteins
/ Research and Analysis Methods
/ Saccharomyces cerevisiae - cytology
/ Saccharomyces cerevisiae - genetics
/ Saccharomyces cerevisiae - metabolism
/ Saccharomyces cerevisiae Proteins - genetics
/ Saccharomyces cerevisiae Proteins - metabolism
/ Tags
/ Yeast
