Taking down the FLAG! How insect cell expression challenges an established tag-system.
In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system.
CSIRO Materials Science and Engineering, Parkville, Victoria, Australia. Peter.Schmidt@CSL.com.au
Peter M
PM
Schmidt
Lindsay G
LG
Sparrow
Rebecca M
RM
Attwood
Xiaowen
X
Xiao
Tim E
TE
Adams
Jennifer L
JL
McKimm-Breschkin
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D000939:Epitopes / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007313:Insecta; D013058:Mass Spectrometry; D009439:Neuraminidase / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase; D009439:Neuraminidase