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Taking down the FLAG! How insect cell expression challenges an established tag-system.

Schmidt PM, Sparrow LG, Attwood RM, Xiao X, Adams TE, McKimm-Breschkin JL - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Materials Science and Engineering, Parkville, Victoria, Australia. Peter.Schmidt@CSL.com.au

ABSTRACT
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.

Show MeSH
Expression and purification of recombinant NA in mammalian cells.Human TB-NA (Hokkaido) fused to the Tetrabrachion stalk (Fig. 1B) was cloned into the pApex-3 vector and expressed in HEK293T cells. Media, flow-through, and purified NA were loaded onto SDS-PAGE and detected by anti-FLAG WB (left panel) or stained by Coomassie (right panel). Secreted FLAG-reactive NA was detected by anti-FLAG WB. Based on the anti-FLAG WB the column flow-through did not contain any residual FLAG reactive NA.
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pone-0037779-g004: Expression and purification of recombinant NA in mammalian cells.Human TB-NA (Hokkaido) fused to the Tetrabrachion stalk (Fig. 1B) was cloned into the pApex-3 vector and expressed in HEK293T cells. Media, flow-through, and purified NA were loaded onto SDS-PAGE and detected by anti-FLAG WB (left panel) or stained by Coomassie (right panel). Secreted FLAG-reactive NA was detected by anti-FLAG WB. Based on the anti-FLAG WB the column flow-through did not contain any residual FLAG reactive NA.

Mentions: As it would be laborious and cost-intensive for many laboratories to replace an established tag system including vectors, antibodies, and affinity matrices we tried to decrease the likelihood of FLAG sulfation. This PTM is catalyzed by the enzyme family of TPSTs which rely on a consensus sequence to identify potential sulfation sites [15]. Although the composition of the consensus sequence is not entirely understood [15], the NA expression vectors were modified to express different residues N-terminal of the FLAG tag to test if FLAG sulfation could be decreased. Changing the sequence AEF-DYKDDDK (Fig. 1A, B) to M-DYKDDDDK (Fig. 1C) did not result in any change of the sulfation ratio for expressed TB-NA (data not shown). However, enzymes expressed with a blunt N-terminal FLAG tag were virtually entirely depleted from the media by anti-FLAG affinity chromatography suggesting that the shortened sequence was not longer recognized as a substrate by TPSTs. In parallel, it was investigated whether the expression of FLAG-tagged NA in a mammalian cell line (HEK 293T) would result in a more favorable ratio of sulfated to non-sulfated FLAG-tagged NA compared to expression in insect cells (BVES). The sequence encoding for the human seasonal TB-N1 was cloned into a mammalian expression vector and expressed in HEK293T cells as described in the Experimental Procedures section. After transient transfections, the media showed strong NA activity and the secreted NA could be detected by anti-FLAG WB (Fig. 4). However, after affinity purification of the FLAG-tagged NA, there was virtually no residual NA detectable in the column flow-through either by activity assays (data not shown) or by anti-FLAG WB (Fig. 4), suggesting purification of the entire FLAG-tagged NA.


Taking down the FLAG! How insect cell expression challenges an established tag-system.

Schmidt PM, Sparrow LG, Attwood RM, Xiao X, Adams TE, McKimm-Breschkin JL - PLoS ONE (2012)

Expression and purification of recombinant NA in mammalian cells.Human TB-NA (Hokkaido) fused to the Tetrabrachion stalk (Fig. 1B) was cloned into the pApex-3 vector and expressed in HEK293T cells. Media, flow-through, and purified NA were loaded onto SDS-PAGE and detected by anti-FLAG WB (left panel) or stained by Coomassie (right panel). Secreted FLAG-reactive NA was detected by anti-FLAG WB. Based on the anti-FLAG WB the column flow-through did not contain any residual FLAG reactive NA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368911&req=5

pone-0037779-g004: Expression and purification of recombinant NA in mammalian cells.Human TB-NA (Hokkaido) fused to the Tetrabrachion stalk (Fig. 1B) was cloned into the pApex-3 vector and expressed in HEK293T cells. Media, flow-through, and purified NA were loaded onto SDS-PAGE and detected by anti-FLAG WB (left panel) or stained by Coomassie (right panel). Secreted FLAG-reactive NA was detected by anti-FLAG WB. Based on the anti-FLAG WB the column flow-through did not contain any residual FLAG reactive NA.
Mentions: As it would be laborious and cost-intensive for many laboratories to replace an established tag system including vectors, antibodies, and affinity matrices we tried to decrease the likelihood of FLAG sulfation. This PTM is catalyzed by the enzyme family of TPSTs which rely on a consensus sequence to identify potential sulfation sites [15]. Although the composition of the consensus sequence is not entirely understood [15], the NA expression vectors were modified to express different residues N-terminal of the FLAG tag to test if FLAG sulfation could be decreased. Changing the sequence AEF-DYKDDDK (Fig. 1A, B) to M-DYKDDDDK (Fig. 1C) did not result in any change of the sulfation ratio for expressed TB-NA (data not shown). However, enzymes expressed with a blunt N-terminal FLAG tag were virtually entirely depleted from the media by anti-FLAG affinity chromatography suggesting that the shortened sequence was not longer recognized as a substrate by TPSTs. In parallel, it was investigated whether the expression of FLAG-tagged NA in a mammalian cell line (HEK 293T) would result in a more favorable ratio of sulfated to non-sulfated FLAG-tagged NA compared to expression in insect cells (BVES). The sequence encoding for the human seasonal TB-N1 was cloned into a mammalian expression vector and expressed in HEK293T cells as described in the Experimental Procedures section. After transient transfections, the media showed strong NA activity and the secreted NA could be detected by anti-FLAG WB (Fig. 4). However, after affinity purification of the FLAG-tagged NA, there was virtually no residual NA detectable in the column flow-through either by activity assays (data not shown) or by anti-FLAG WB (Fig. 4), suggesting purification of the entire FLAG-tagged NA.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Materials Science and Engineering, Parkville, Victoria, Australia. Peter.Schmidt@CSL.com.au

ABSTRACT
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.

Show MeSH