Abstract
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.
Highlights
Protein tags are peptide sequences that can be grafted onto a target recombinant protein
Cells were maintained at 37 ̊C and 5% CO2 in a humidified atmosphere. pPB-FRT-PGKp-BSD-mEGFP-FLAG-CD55 (C81A) and pCMVhyPBase [20] were cotransfected into HEK293 WT cells and selected with 10 μg/ml blasticidin for stable expression
We tried to identify factors that are involved in the retention of misfolded GPI-anchored proteins (GPI-APs) in the endoplasmic reticulum (ER) using the genome-scale CRISPR-Cas9 knockout (GeCKO) library [21] (Fig 1A)
Summary
Protein tags are peptide sequences that can be grafted onto a target recombinant protein. Protein tags can be removed by chemical agents or by enzymatic reactions. A FLAG tag is a commonly and widely used protein tag that could be added either to the N-terminus, C-terminus or intermediate regions of the targeted protein. Some types of commercially available antibodies (e.g., M1/4E11) recognize the epitope only when it is present at the N-terminus. Other available antibodies (e.g., M2) show no position sensitivity. Several studies have reported that FLAG is not a good protein tag because a tyrosine residue on the FLAG-tag can be sulfated [1, 2]. For example, M2, seem sensitive to the sulfation state of the FLAG tag [2, 3]
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