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 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.