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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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Expression and purification of recombinant NA in insect cells.The expression and secretion of GCN-pLI and TB-based soluble Hokkaido NA was monitored by anti-FLAG WB (2A, B) and by NA activity assays (2C). The gel filtration chromatogram for both NA constructs is shown in 2D. 2E shows the processed insect cell media before and after anti-FLAG affinity purification in a Coomassie stained gel (2E left panel) and corresponding WB (2E right panel). Figure 2F shows the FLAG- and Lectin/IEX-purified pN1/2009 NA loaded onto an SDS-PAGE and stained by Coomassie (2F left panel) or detected by anti-FLAG WB (2F right panel). The amino acid sequence obtained by N-terminal sequencing of both enzymes is shown in 2G.
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pone-0037779-g002: Expression and purification of recombinant NA in insect cells.The expression and secretion of GCN-pLI and TB-based soluble Hokkaido NA was monitored by anti-FLAG WB (2A, B) and by NA activity assays (2C). The gel filtration chromatogram for both NA constructs is shown in 2D. 2E shows the processed insect cell media before and after anti-FLAG affinity purification in a Coomassie stained gel (2E left panel) and corresponding WB (2E right panel). Figure 2F shows the FLAG- and Lectin/IEX-purified pN1/2009 NA loaded onto an SDS-PAGE and stained by Coomassie (2F left panel) or detected by anti-FLAG WB (2F right panel). The amino acid sequence obtained by N-terminal sequencing of both enzymes is shown in 2G.

Mentions: In order to obtain purified neuraminidase (NA) for biochemical characterization and crystallization studies human N1 NA containing the artificial GCN-pLI or the Tetrabrachion stalks (Fig. 1A, B) were expressed as described earlier [9]. Both insect cell expressions showed maximum NA secretion 84 h post infection without visible degradation products as judged by anti-FLAG western blot (WB; Fig. 2A, B). The Tetrabrachion-based construct (Fig. 2B) resulted in higher yields in agreement with the corresponding NA activity assays (Fig. 2C) which showed approximately four-fold higher NA activity for the Tetrabrachion-based NA compared to the GCN-pLI-NA. The higher expression levels of the TB-based NA-construct as well as its higher molecular weight were corroborated by gel filtration chromatography showing a four-fold higher absorption and faster elution compared to GCN-pLI-NA (Fig. 2D). Both expressions resulted in highly pure NA with no visible contaminating proteins as judged by SDS-PAGE (Fig. 2E left panel) and anti-FLAG WB (Fig. 2E right panel). The flow-through after anti-FLAG affinity purification showed no signal in the anti-FLAG WB suggesting that the entire FLAG-reactive NA has been purified from the media in a single run (Fig. 2E, right panel). Surprisingly, when the flow-throughs were checked for residual NA activity it became evident that 49% of the activity of the GCN-pLI-based enzyme and 84% of the Tetrabrachion-based NA (data not shown) were still in the flow-through despite the results of the WB suggesting the entire depletion of both expressed enzymes. Similar results were obtained for the TB-based pN1/2009 construct (Fig. 1C; data not shown).


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 insect cells.The expression and secretion of GCN-pLI and TB-based soluble Hokkaido NA was monitored by anti-FLAG WB (2A, B) and by NA activity assays (2C). The gel filtration chromatogram for both NA constructs is shown in 2D. 2E shows the processed insect cell media before and after anti-FLAG affinity purification in a Coomassie stained gel (2E left panel) and corresponding WB (2E right panel). Figure 2F shows the FLAG- and Lectin/IEX-purified pN1/2009 NA loaded onto an SDS-PAGE and stained by Coomassie (2F left panel) or detected by anti-FLAG WB (2F right panel). The amino acid sequence obtained by N-terminal sequencing of both enzymes is shown in 2G.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037779-g002: Expression and purification of recombinant NA in insect cells.The expression and secretion of GCN-pLI and TB-based soluble Hokkaido NA was monitored by anti-FLAG WB (2A, B) and by NA activity assays (2C). The gel filtration chromatogram for both NA constructs is shown in 2D. 2E shows the processed insect cell media before and after anti-FLAG affinity purification in a Coomassie stained gel (2E left panel) and corresponding WB (2E right panel). Figure 2F shows the FLAG- and Lectin/IEX-purified pN1/2009 NA loaded onto an SDS-PAGE and stained by Coomassie (2F left panel) or detected by anti-FLAG WB (2F right panel). The amino acid sequence obtained by N-terminal sequencing of both enzymes is shown in 2G.
Mentions: In order to obtain purified neuraminidase (NA) for biochemical characterization and crystallization studies human N1 NA containing the artificial GCN-pLI or the Tetrabrachion stalks (Fig. 1A, B) were expressed as described earlier [9]. Both insect cell expressions showed maximum NA secretion 84 h post infection without visible degradation products as judged by anti-FLAG western blot (WB; Fig. 2A, B). The Tetrabrachion-based construct (Fig. 2B) resulted in higher yields in agreement with the corresponding NA activity assays (Fig. 2C) which showed approximately four-fold higher NA activity for the Tetrabrachion-based NA compared to the GCN-pLI-NA. The higher expression levels of the TB-based NA-construct as well as its higher molecular weight were corroborated by gel filtration chromatography showing a four-fold higher absorption and faster elution compared to GCN-pLI-NA (Fig. 2D). Both expressions resulted in highly pure NA with no visible contaminating proteins as judged by SDS-PAGE (Fig. 2E left panel) and anti-FLAG WB (Fig. 2E right panel). The flow-through after anti-FLAG affinity purification showed no signal in the anti-FLAG WB suggesting that the entire FLAG-reactive NA has been purified from the media in a single run (Fig. 2E, right panel). Surprisingly, when the flow-throughs were checked for residual NA activity it became evident that 49% of the activity of the GCN-pLI-based enzyme and 84% of the Tetrabrachion-based NA (data not shown) were still in the flow-through despite the results of the WB suggesting the entire depletion of both expressed enzymes. Similar results were obtained for the TB-based pN1/2009 construct (Fig. 1C; data not shown).

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