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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.

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Identification of FLAG-tag tyrosine-sulfation.Lectin purified TB-Hokkaido NA was incubated with different PTM-cleaving enzymes. The anti-FLAG WB is shown in Fig. 3A. Addition of sulfatase made the FLAG tag accessible to the anti-FLAG mAb. Desulfation kinetics of Lectin/IEX-purified pN1/2009 NA using sulfatase type VIII (Abalone entrails) are shown in 3B. Activity of all 4 sulfatases tested was efficiently inhibited by the addition of vanadate-phenyl-Ester (3C). Fig. 3D shows the negative linear mode MS of the sulfated control peptide CCK-8 at 1143.5 Da and its non-sulfated form at 1063.3 Da (−80 Da). The Thrombin cleaved FLAG tag was detected in its sulfated and non-sulfated form at a molecular weight of 1690.9 and 1611.1 Da (−80 Da), respectively. Fig. 3E shows the same sample measured in positive linear mode. Only the non-sulfated peptides (−80 Da) were detected with an additional weight due to protonation (+1 Da). Fig. 3F shows the WB of FLAG- and Lectin-purified NA (pN1/2009) detected with different anti-FLAG antibodies as well as the Coomassie stained gel of the same samples.
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pone-0037779-g003: Identification of FLAG-tag tyrosine-sulfation.Lectin purified TB-Hokkaido NA was incubated with different PTM-cleaving enzymes. The anti-FLAG WB is shown in Fig. 3A. Addition of sulfatase made the FLAG tag accessible to the anti-FLAG mAb. Desulfation kinetics of Lectin/IEX-purified pN1/2009 NA using sulfatase type VIII (Abalone entrails) are shown in 3B. Activity of all 4 sulfatases tested was efficiently inhibited by the addition of vanadate-phenyl-Ester (3C). Fig. 3D shows the negative linear mode MS of the sulfated control peptide CCK-8 at 1143.5 Da and its non-sulfated form at 1063.3 Da (−80 Da). The Thrombin cleaved FLAG tag was detected in its sulfated and non-sulfated form at a molecular weight of 1690.9 and 1611.1 Da (−80 Da), respectively. Fig. 3E shows the same sample measured in positive linear mode. Only the non-sulfated peptides (−80 Da) were detected with an additional weight due to protonation (+1 Da). Fig. 3F shows the WB of FLAG- and Lectin-purified NA (pN1/2009) detected with different anti-FLAG antibodies as well as the Coomassie stained gel of the same samples.

Mentions: These results narrowed down potential explanations for the non-reactivity of the tag to unfavorable folding or post-translational modification of the tag. As the Tetrabrachion stalk has been described as extremely rigid [11], [12] an accidental folding of the highly soluble FLAG tag masking the epitope in only a certain subpopulation of the expressed protein appeared rather unlikely. In contrast, preliminary in-silico prediction suggested a potential phosphorylation or sulfation of the FLAG tyrosine as well as a glycosylation of an adjacent asparagine residue (www.expasy.ch). From these predictions, tyrosine sulfation appeared to be most likely as this PTM is a known modification of secreted proteins [13], [14] and it requires a high density of negative charged residues in close proximity to the tyrosine (a comprehensive review on the characteristics of the sulfation consensus site can be found here [15]). To test these predictions, non-FLAG reactive TB-N1 (Hokkaido) was incubated with λ-phosphatase, H1 sulfatase, or PNGaseF and subsequently subjected to anti-FLAG WB analysis. The results of these blots showed clearly that incubation with sulfatase but not with phosphatase or PNGaseF restored the reactivity of the FLAG epitope for the anti-FLAG antibody (Fig. 3A) suggesting that indeed tyrosine-sulfation of the FLAG tag was responsible for masking the FLAG epitope. Subsequent experiments showed that desulfation of the FLAG tag could be accomplished by different sulfatases including type H1 (Helix pomatia), type IV and V (Patella vulgata) and type VIII (Abalone entrails). All four sulfatases tested were efficiently blocked by the potent sulfatase inhibitor vanadate-phenyl-ester [16] underlining the specificity of the desulfation reaction (Fig. 3C). In addition the time-dependency of the desulfation reaction was shown (Fig. 3B).


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)

Identification of FLAG-tag tyrosine-sulfation.Lectin purified TB-Hokkaido NA was incubated with different PTM-cleaving enzymes. The anti-FLAG WB is shown in Fig. 3A. Addition of sulfatase made the FLAG tag accessible to the anti-FLAG mAb. Desulfation kinetics of Lectin/IEX-purified pN1/2009 NA using sulfatase type VIII (Abalone entrails) are shown in 3B. Activity of all 4 sulfatases tested was efficiently inhibited by the addition of vanadate-phenyl-Ester (3C). Fig. 3D shows the negative linear mode MS of the sulfated control peptide CCK-8 at 1143.5 Da and its non-sulfated form at 1063.3 Da (−80 Da). The Thrombin cleaved FLAG tag was detected in its sulfated and non-sulfated form at a molecular weight of 1690.9 and 1611.1 Da (−80 Da), respectively. Fig. 3E shows the same sample measured in positive linear mode. Only the non-sulfated peptides (−80 Da) were detected with an additional weight due to protonation (+1 Da). Fig. 3F shows the WB of FLAG- and Lectin-purified NA (pN1/2009) detected with different anti-FLAG antibodies as well as the Coomassie stained gel of the same samples.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037779-g003: Identification of FLAG-tag tyrosine-sulfation.Lectin purified TB-Hokkaido NA was incubated with different PTM-cleaving enzymes. The anti-FLAG WB is shown in Fig. 3A. Addition of sulfatase made the FLAG tag accessible to the anti-FLAG mAb. Desulfation kinetics of Lectin/IEX-purified pN1/2009 NA using sulfatase type VIII (Abalone entrails) are shown in 3B. Activity of all 4 sulfatases tested was efficiently inhibited by the addition of vanadate-phenyl-Ester (3C). Fig. 3D shows the negative linear mode MS of the sulfated control peptide CCK-8 at 1143.5 Da and its non-sulfated form at 1063.3 Da (−80 Da). The Thrombin cleaved FLAG tag was detected in its sulfated and non-sulfated form at a molecular weight of 1690.9 and 1611.1 Da (−80 Da), respectively. Fig. 3E shows the same sample measured in positive linear mode. Only the non-sulfated peptides (−80 Da) were detected with an additional weight due to protonation (+1 Da). Fig. 3F shows the WB of FLAG- and Lectin-purified NA (pN1/2009) detected with different anti-FLAG antibodies as well as the Coomassie stained gel of the same samples.
Mentions: These results narrowed down potential explanations for the non-reactivity of the tag to unfavorable folding or post-translational modification of the tag. As the Tetrabrachion stalk has been described as extremely rigid [11], [12] an accidental folding of the highly soluble FLAG tag masking the epitope in only a certain subpopulation of the expressed protein appeared rather unlikely. In contrast, preliminary in-silico prediction suggested a potential phosphorylation or sulfation of the FLAG tyrosine as well as a glycosylation of an adjacent asparagine residue (www.expasy.ch). From these predictions, tyrosine sulfation appeared to be most likely as this PTM is a known modification of secreted proteins [13], [14] and it requires a high density of negative charged residues in close proximity to the tyrosine (a comprehensive review on the characteristics of the sulfation consensus site can be found here [15]). To test these predictions, non-FLAG reactive TB-N1 (Hokkaido) was incubated with λ-phosphatase, H1 sulfatase, or PNGaseF and subsequently subjected to anti-FLAG WB analysis. The results of these blots showed clearly that incubation with sulfatase but not with phosphatase or PNGaseF restored the reactivity of the FLAG epitope for the anti-FLAG antibody (Fig. 3A) suggesting that indeed tyrosine-sulfation of the FLAG tag was responsible for masking the FLAG epitope. Subsequent experiments showed that desulfation of the FLAG tag could be accomplished by different sulfatases including type H1 (Helix pomatia), type IV and V (Patella vulgata) and type VIII (Abalone entrails). All four sulfatases tested were efficiently blocked by the potent sulfatase inhibitor vanadate-phenyl-ester [16] underlining the specificity of the desulfation reaction (Fig. 3C). In addition the time-dependency of the desulfation reaction was shown (Fig. 3B).

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