Stoib Anna, Shojaei Sahar, Siligan Christine, Horner Andreas
Institute of Biophysics Johannes Kepler University Linz Gruberstr. 40 4020 Linz Austria.
Small Sci. 2025 Jan 27;5(5):2400571. doi: 10.1002/smsc.202400571. eCollection 2025 May.
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 UreI, an acid-gated urea channel of . 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 UreI 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.
亲和标签是蛋白质纯化中的关键组成部分。尽管有多项迹象表明它们会影响蛋白质的结构和功能，但这种影响往往不为人知或被忽视。这在体外数据的解释中不必要地引入了模糊性。为了说明这一点，尿素和氨酵母互补分析被用作一种筛选工具，以评估与野生型蛋白相比，各种亲和标签位置的功能差异，使用的是嗜热栖热菌的酸门控尿素通道UreI。酵母互补分析通过观察生长情况来测试缺失菌株中表达的外源蛋白的pH依赖性功能。如果外源蛋白能够替代缺失的内源蛋白的功能，酵母细胞就能在特定的分析条件下生长。标签的整体位置，甚至标签切割后少量的N端或C端残留氨基酸都会对UreI功能产生溶质特异性影响，这表明存在一种复杂的溶质选择性机制，并强调了体内表征的必要性。这种经济高效的酵母互补分析可用于测试多种溶质。它可以用作在进行定量体外蛋白质表征之前，对亲和标签位置或蛋白质突变进行初步筛选的工具。
