Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH.

酵母展示已成为现代生物技术的重要工具，具有用于真核蛋白质工程的许多优势。基于抗体的肽相互作用通常用于量化酵母表面表达（例如，通过将目标蛋白融合到 FLAG、Myc、多组氨酸或其他肽标签）。然而，抗体-抗原相互作用需要高稳定性才能准确定量，基于这种相互作用的传统标签系统可能与低 pH 环境不兼容。在这项研究中，将 SNAP 标签引入酵母展示平台，以规避传统抗体展示标签在低 pH 值下的缺点。SNAP 与其小分子底物形成共价键，从而实现精确且不依赖 pH 值的蛋白质显示标记。我们将 SNAP 标签与传统的基于抗体的肽融合以及使用抗体片段可结晶 (Fc) 基因库作为低 pH 蛋白质工程案例研究的直接荧光域融合进行了比较。我们的结果表明，共价 SNAP 标签可以有效地量化低 pH 下的蛋白质表面表达，从而能够富集在 pH 6.0 时对新生儿 Fc 受体 (FcRn) 亲和力增加的 Fc 变体。因此，共价 SNAP 标签的结合克服了传统基于抗体的表达标签的缺点，并使蛋白质工程应用能够在生理 pH 值之外进行。能够富集在 pH 6.0 时对新生儿 Fc 受体 (FcRn) 具有更高亲和力的 Fc 变体。因此，共价 SNAP 标签的结合克服了传统基于抗体的表达标签的缺点，并使蛋白质工程应用能够在生理 pH 值之外进行。能够富集在 pH 6.0 时对新生儿 Fc 受体 (FcRn) 具有更高亲和力的 Fc 变体。因此，共价 SNAP 标签的结合克服了传统基于抗体的表达标签的缺点，并使蛋白质工程应用能够在生理 pH 值之外进行。