This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (K_D) of select WW-YAP-binding peptides: K_D values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22⁺ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.

中文翻译：

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使用目标展示磁化酵母筛选 mRNA 展示肽文库以鉴定配体

这项工作首次使用酵母展示的蛋白质靶标来筛选环状和线性肽的 mRNA 展示文库。采用 Yes 相关蛋白 1 (WW-YAP) 的 WW 结构域和线粒体输入受体亚基 TOM22 作为蛋白靶点。用氧化铁纳米颗粒磁化展示 WW-YAP 或 TOM22 的酵母细胞，以实现靶标结合 mRNA-肽融合体的分离。进行平衡吸附研究以估计所选 WW-YAP 结合肽的结合亲和力 ( _KD )：环_{[ M-AFRLC-K] 及其线性同源物的}KD值为 37 和 4 μM，40 和环[M-LDFVNHRSRG-K]及其线性同源物分别为3 μM。TOM22 结合肽环[M-PELNRAI-K] 与磁珠缀合，并与表达 TOM22 和荧光素酶的酵母细胞一起孵育。基于荧光素酶的检测显示，与对照细胞相比， TOM22 ⁺酵母的结合力高 4.5 倍。这项工作表明，整合 mRNA 和酵母展示可加速肽配体的发现。