When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.

中文翻译：

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筛选针对磁化酵母细胞靶标的酵母展示文库可以有效分离膜蛋白结合剂。

当从酵母展示的组合文库中分离结合物时，通常利用靶蛋白的可溶的，重组表达的形式。作为替代方案，我们描述了在磁化酵母细胞上以表面融合体形式显示的靶蛋白的用途。在我们的策略中，目标蛋白与氧化铁结合蛋白在酵母表面共表达。将这些酵母细胞与氧化铁纳米颗粒一起孵育会导致其磁化。随后，可以使用磁体分离与磁化的靶细胞相互作用的结合细胞。使用已知的具有适度结合亲和力的结合物-靶对（KD≈400 nM），我们显示了在单轮筛选中，低频存在的结合物（105中的1）可以富集100倍以上，这表明可行性筛选组合库。随后，我们筛选了Sso7d酵母展示文库和针对酵母展示靶标的纳米抗体变异体，以分离特定于线粒体膜蛋白TOM22（KD≈272-1934 nM）的胞质结构域和c-Kit受体的胞外结构域（KD≈93）的结合剂到KD> 2000 nM）。进一步的研究表明，使用这种方法鉴定出的TOM22结合物可用于从细胞裂解物中富集线粒体，从而确认与天然线粒体蛋白质的结合。与重组可溶性表达相反，表达膜蛋白或其结构域作为酵母细胞表面融合的容易性使得使用酵母展示的靶标特别有吸引力。因此，我们预计使用磁化酵母细胞靶标将能够有效分离膜蛋白的结合剂。