Zajc Charlotte U, Sylvander Elise, Teufl Magdalena, Traxlmayr Michael W
Department of Chemistry, Institute of Biochemistry, BOKU University; CD Laboratory for Next Generation CAR T Cells;
CD Laboratory for Next Generation CAR T Cells; St. Anna Children's Cancer Research Institute, CCRI.
J Vis Exp. 2024 Nov 29(213). doi: 10.3791/66994.
Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.
蛋白质工程能够改进给定蛋白质的现有功能或产生新功能。酵母表面展示是蛋白质工程领域应用最广泛、用途最多样的工具之一，在酵母表面表达一组随机化的蛋白质。表型（例如，酵母展示的蛋白质与目标抗原的结合）与基因型（编码蛋白质变体的质粒）的关联使得能够针对所需特性对该文库进行筛选，并对富集的变体进行后续测序。通过将磁珠筛选与流式细胞术分选相结合，可以筛选和富集与目标抗原结合增强的蛋白质变体。值得注意的是，除了亲和力成熟外，在没有任何初始结合亲和力的情况下也能实现与目标的结合。在这里，我们提供了一个分步方案，涵盖酵母表面展示筛选活动的所有关键部分，并给出典型酵母表面展示结果的示例。我们证明酵母表面展示是一种广泛适用且稳健的方法，任何能够使用流式细胞术的分子生物学实验室都可以采用。
