A method for labeling proteins with tags at the native genomic loci in budding yeast.
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 limit...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5411076?pdf=render |
| _version_ | 1818081906964561920 |
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| author | Qian Wang Huijun Xue Siqi Li Ying Chen Xuelei Tian Xin Xu Wei Xiao Yu Vincent Fu |
| author_facet | Qian Wang Huijun Xue Siqi Li Ying Chen Xuelei Tian Xin Xu Wei Xiao Yu Vincent Fu |
| author_sort | Qian Wang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T19:13:39Z |
| format | Article |
| id | doaj.art-ba3a1bf697694647baa1ffe981ff452d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T19:13:39Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ba3a1bf697694647baa1ffe981ff452d2022-12-22T01:36:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01125e017618410.1371/journal.pone.0176184A method for labeling proteins with tags at the native genomic loci in budding yeast.Qian WangHuijun XueSiqi LiYing ChenXuelei TianXin XuWei XiaoYu Vincent FuFluorescent 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.http://europepmc.org/articles/PMC5411076?pdf=render |
| spellingShingle | Qian Wang Huijun Xue Siqi Li Ying Chen Xuelei Tian Xin Xu Wei Xiao Yu Vincent Fu A method for labeling proteins with tags at the native genomic loci in budding yeast. PLoS ONE |
| title | A method for labeling proteins with tags at the native genomic loci in budding yeast. |
| title_full | A method for labeling proteins with tags at the native genomic loci in budding yeast. |
| title_fullStr | A method for labeling proteins with tags at the native genomic loci in budding yeast. |
| title_full_unstemmed | A method for labeling proteins with tags at the native genomic loci in budding yeast. |
| title_short | A method for labeling proteins with tags at the native genomic loci in budding yeast. |
| title_sort | method for labeling proteins with tags at the native genomic loci in budding yeast |
| url | http://europepmc.org/articles/PMC5411076?pdf=render |
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