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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Autores principales:	Xin-Yu Guo, Xiao-Dong Gao, Morihisa Fujita
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9a25ed81827643de90cd70a9dda83952
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spelling	oai:doaj.org-article:9a25ed81827643de90cd70a9dda839522021-11-25T05:54:22ZSulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.1932-620310.1371/journal.pone.0250805https://doaj.org/article/9a25ed81827643de90cd70a9dda839522021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0250805https://doaj.org/toc/1932-6203A 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.Xin-Yu GuoXiao-Dong GaoMorihisa FujitaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 5, p e0250805 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.
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
author	Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita
author_facet	Xin-Yu Guo Xiao-Dong Gao Morihisa Fujita
author_sort	Xin-Yu Guo
title	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_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_sort	sulfation of a flag tag mediated by slc35b2 and tpst2 affects antibody recognition.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/9a25ed81827643de90cd70a9dda83952
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Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition.

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