The accurate transmission of signals by the canonical ERK1/2 kinase pathway critically relies on the proper assembly of an intricate multiprotein complex by the scaffold protein Shoc2. However, the details of the mechanism by which Shoc2 guides ERK1/2 signals are not clear, in part, due to the lack of research tools targeting specific protein binding moieties of Shoc2. We report generation and characterization of single domain antibodies against human Shoc2 using a universal synthetic library of humanized nanobodies. Our results identify eight synthetic single-domain antibodies and show that two evaluated antibodies have binding affinities to Shoc2 in the nanomolar range. High affinity antibodies were uniquely suited for the analysis of the Shoc2 complex assembly. Selected single-domain antibodies were also functional in intracellular assays. This study illustrates that Shoc2 single-domain antibodies can be used to understand functional mechanisms governing complex multiprotein signaling modules and have promise in application for therapies that require modulation of the ERK1/2-associated diseases.

中文翻译：

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用于信号传导支架Shoc2功能靶向的单域抗体。

通过经典的ERK1 / 2激酶途径的信号的准确传输至关重要地依赖于支架蛋白Shoc2对复杂的多蛋白复合物的正确组装。但是，Shoc2指导ERK1 / 2信号的机制的细节尚不清楚，部分原因是缺乏针对Shoc2特定蛋白结合部分的研究工具。我们报告了使用人源化纳米抗体的通用合成库针对人类Shoc2的单域抗体的生成和表征。我们的结果鉴定出八种合成的单域抗体，并显示两种评估的抗体在纳摩尔范围内对Shoc2具有结合亲和力。高亲和力抗体特别适合于Shoc2复合物装配体的分析。所选的单结构域抗体在细胞内测定中也起作用。