Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low μM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

中文翻译：

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由噬菌体展示选择的反平行α螺旋和β折叠组成的蛋白质-蛋白质结合表面的实用性


在过去的三十年中，各种各样的小蛋白质结构域被用作支架，利用各种蛋白质展示和富集技术生成通用蛋白质结合试剂。为了扩展可能用于此目的的支架和蛋白质表面的全部功能，我们探索了（i）小型高度二硫键键合的 4 螺旋束中的一对反平行 α 螺旋的效用，即来自回复的 CC4 结构域-诱导富含半胱氨酸的蛋白质，具有 Kazal 基序和 (ii) 凹形 β 折叠表面和人类 FN3 结构域中的两个相邻环，这是广泛使用的单体平台的支架。使用 M13 噬菌体展示和下一代测序，我们观察到，在两个系统中，大约 3000 万个变体的文库包含对与多种哺乳动物蛋白的胞外域相对应的诱饵具有低 μM 范围的亲和力的结合蛋白。基于 CC4 和 FN3 的结合蛋白融合至 Fc 结构域的 N 和/或 C 末端，并用于转染细胞的免疫染色。此外，基于 FN3 的结合蛋白被插入 AAV 的 VP1 中，以引导 AAV 感染表达特定表面受体的细胞。最后，基于 FN3 的结合蛋白被插入细胞外收缩注射系统颗粒的 Pvc13 尾纤维蛋白中，以指导蛋白质货物递送至表达特定表面受体的细胞。这些实验支持 CC4 螺旋 B 和 C 以及 FN3 β 链 C、D 和 F 以及相邻环 CD 和 FG 作为工程通用蛋白质结合试剂的表面的用途。