Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.
A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface....
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| Language: | English |
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Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0250805&type=printable |
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| author | Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita |
| author_facet | Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita |
| author_sort | Xin-Yu Guo |
| collection | DOAJ |
| description | A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3'-phosphoadenosine-5'-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway. |
| format | Article |
| id | doaj-art-9a25ed81827643de90cd70a9dda83952 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-9a25ed81827643de90cd70a9dda839522025-08-20T02:55:27ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01165e025080510.1371/journal.pone.0250805Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.Xin-Yu GuoXiao-Dong GaoMorihisa FujitaA FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3'-phosphoadenosine-5'-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0250805&type=printable |
| spellingShingle | Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. PLoS ONE |
| title | Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. |
| title_full | Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. |
| title_fullStr | Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. |
| title_full_unstemmed | Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. |
| title_short | Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition. |
| title_sort | sulfation of a flag tag mediated by slc35b2 and tpst2 affects antibody recognition |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0250805&type=printable |
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