Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori
Affinity tags are a crucial component in protein purification. Despite several indications that they can influence protein structure and function, this influence is often unknown or disregarded. This unnecessarily introduces ambiguity in the interpretation of in vitro data. To illustrate that, urea...
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Wiley-VCH
2025-05-01
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| Series: | Small Science |
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| Online Access: | https://doi.org/10.1002/smsc.202400571 |
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| author | Anna Stoib Sahar Shojaei Christine Siligan Andreas Horner |
| author_facet | Anna Stoib Sahar Shojaei Christine Siligan Andreas Horner |
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| description | Affinity tags are a crucial component in protein purification. Despite several indications that they can influence protein structure and function, this influence is often unknown or disregarded. This unnecessarily introduces ambiguity in the interpretation of in vitro data. To illustrate that, urea and ammonia yeast complementation assays are used as a screening tool to assess functional differences in various affinity tag positions, compared to the WT protein, using HpUreI, an acid‐gated urea channel of Helicobacter pylori. Yeast complementation assays test the pH‐dependent functionality of exogenous proteins expressed in deletion strains by observing growth. If the exogenous protein is able to replace the function of the deleted endogenous protein, yeast cells can demonstrate growth under specific assay conditions. The overall tag position and even a minor amount of residual N‐ or C‐terminal amino acids following tag cleavage exert a solute‐specific influence on HpUreI functionality, suggesting a complex solute selectivity mechanism and underscores the necessity for in vivo characterization. This cost‐effective yeast complementation assay can be adapted to test a broad range of solutes. It can be used as a preliminary screening tool for affinity tag positions or protein mutations before quantitative in vitro protein characterization. |
| format | Article |
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| institution | Kabale University |
| issn | 2688-4046 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Wiley-VCH |
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| series | Small Science |
| spelling | doaj-art-52f9e383fdb948b7b6cd2f876761a4602025-08-20T03:52:29ZengWiley-VCHSmall Science2688-40462025-05-0155n/an/a10.1002/smsc.202400571Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pyloriAnna Stoib0Sahar Shojaei1Christine Siligan2Andreas Horner3Institute of Biophysics Johannes Kepler University Linz Gruberstr. 40 4020 Linz AustriaInstitute of Biophysics Johannes Kepler University Linz Gruberstr. 40 4020 Linz AustriaInstitute of Biophysics Johannes Kepler University Linz Gruberstr. 40 4020 Linz AustriaInstitute of Biophysics Johannes Kepler University Linz Gruberstr. 40 4020 Linz AustriaAffinity tags are a crucial component in protein purification. Despite several indications that they can influence protein structure and function, this influence is often unknown or disregarded. This unnecessarily introduces ambiguity in the interpretation of in vitro data. To illustrate that, urea and ammonia yeast complementation assays are used as a screening tool to assess functional differences in various affinity tag positions, compared to the WT protein, using HpUreI, an acid‐gated urea channel of Helicobacter pylori. Yeast complementation assays test the pH‐dependent functionality of exogenous proteins expressed in deletion strains by observing growth. If the exogenous protein is able to replace the function of the deleted endogenous protein, yeast cells can demonstrate growth under specific assay conditions. The overall tag position and even a minor amount of residual N‐ or C‐terminal amino acids following tag cleavage exert a solute‐specific influence on HpUreI functionality, suggesting a complex solute selectivity mechanism and underscores the necessity for in vivo characterization. This cost‐effective yeast complementation assay can be adapted to test a broad range of solutes. It can be used as a preliminary screening tool for affinity tag positions or protein mutations before quantitative in vitro protein characterization.https://doi.org/10.1002/smsc.202400571affinity tag positionsHelicobacter pyloriHp UreIpH‐gated urea channelsyeast complementation assays |
| spellingShingle | Anna Stoib Sahar Shojaei Christine Siligan Andreas Horner Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori Small Science affinity tag positions Helicobacter pylori Hp UreI pH‐gated urea channels yeast complementation assays |
| title | Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori |
| title_full | Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori |
| title_fullStr | Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori |
| title_full_unstemmed | Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori |
| title_short | Yeast Complementation Assays Demonstrating the Importance of the Affinity Tag Position in Membrane Protein Purification, as Exemplified by HpUreI, the pH‐Gated Urea Channel of Helicobacter pylori |
| title_sort | yeast complementation assays demonstrating the importance of the affinity tag position in membrane protein purification as exemplified by hpurei the ph gated urea channel of helicobacter pylori |
| topic | affinity tag positions Helicobacter pylori Hp UreI pH‐gated urea channels yeast complementation assays |
| url | https://doi.org/10.1002/smsc.202400571 |
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