Yeast surface display selections against mammalian cell monolayers have proven effective in isolating proteins with novel binding activity. Recent advances in this technique allow for the recovery of clones with even micromolar binding affinities. However, no efficient method has been shown for affinity-based selection in this context. This study demonstrates the effectiveness of titratable avidity reduction using dithiothreitol to achieve this goal. A series of epidermal growth factor receptor binding fibronectin domains with a range of affinities are used to quantitatively identify the number of ligands per yeast cell that yield the strongest selectivity between strong, moderate, and weak affinities. Notably, reduction of ligand display to 3,000-6,000 ligands per yeast cell of a 2 nM binder yields 16-fold better selectivity than that to a 17 nM binder. These lessons are applied to affinity maturation of an EpCAM-binding fibronectin population, yielding an enriched pool of ligands with significantly stronger affinity than that of an analogous pool sorted by standard cellular selection methods. Collectively, this study offers a facile approach for affinity selection of yeast-displayed ligands against full-length cellular targets and demonstrates the effectiveness of this method by generating EpCAM-binding ligands that are promising for further applications.

中文翻译：

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可滴定亲和力降低增强了酵母展示配体的哺乳动物细胞选择中的亲和力区分。

已证明针对哺乳动物细胞单层的酵母表面展示选择可有效分离具有新结合活性的蛋白质。该技术的最新进展允许回收甚至具有微摩尔结合亲和力的克隆。但是，在这种情况下，尚未显示有效的方法用于基于亲和力的选择。这项研究证明了使用二硫苏糖醇降低可滴定亲和力的有效性，以实现这一目标。具有一系列亲和力的一系列表皮生长因子受体结合纤连蛋白结构域用于定量鉴定每个酵母细胞中产生强，中和弱亲和力的最强选择性的配体数量。值得注意的是，配体展示减少到3,000-6，每个酵母细胞具有2 nM结合剂的000个配体，其选择性比对17 nM结合剂的选择性高16倍。这些课程适用于结合EpCAM的纤连蛋白群体的亲和力成熟，产生的配体富集池比通过标准细胞选择方法分选的类似池具有更强的亲和力。总的来说，这项研究为酵母展示的配体对全长细胞靶标的亲和力选择提供了一种简便的方法，并通过产生有希望进一步应用的EpCAM结合配体证明了该方法的有效性。产生的配体富集池比通过标准细胞选择方法分选的类似池具有更强的亲和力。总的来说，这项研究为酵母展示的配体对全长细胞靶标的亲和力选择提供了一种简便的方法，并通过产生有希望进一步应用的EpCAM结合配体证明了该方法的有效性。产生的配体富集池比通过标准细胞选择方法分选的类似池具有更强的亲和力。总的来说，这项研究为酵母展示的配体对全长细胞靶标的亲和力选择提供了一种简便的方法，并通过产生有希望进一步应用的EpCAM结合配体证明了该方法的有效性。