We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

中文翻译：

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通过核糖体展示和蛋白质原位固定检测蛋白质-蛋白质相互作用

我们描述了一种通过结合两种无细胞蛋白质技术（即核糖体展示和蛋白质原位固定化）来鉴定蛋白质-蛋白质相互作用的方法。该方法只需要 PCR 片段作为起始材料，目标蛋白质是通过无细胞蛋白质合成制成的，要么与它们的编码 mRNA 相关联作为核糖体复合物，要么固定在固体表面上。核糖体复合物的使用允许从它们附着的编码 mRNA 中识别相互作用的蛋白质伙伴。为了演示这些程序，我们采用了淋巴细胞信号蛋白 Vav1 和 Grb2，并确认了 Grb2 与 Vav1 的 N 端 SH3 域之间的相互作用。该方法有望用于成对蛋白质相互作用的库筛选，直至单个域或基序映射的分析水平。