Tonikian Raffi, Zhang Yingnan, Boone Charles, Sidhu Sachdev S
Department of Molecular and Medical Genetics, University of Toronto, #4398 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Nat Protoc. 2007;2(6):1368-86. doi: 10.1038/nprot.2007.151.
Signaling complexes usually involve multidomain proteins containing catalytic domains and peptide recognition modules (PRMs), which mediate protein-protein interactions and assemble complexes by binding to ligands containing a core sequence motif. Concomitant to large-scale physical interaction screening, considerable effort has been devoted toward the elucidation of consensus profiles for common PRMs. We describe herein a robust and proven protocol to generate consensus profiles for PRMs using phage-displayed peptide libraries. The initial phase of the protocol entails the cloning, expression and purification of PRMs as fusion proteins, in addition to the construction of highly diverse phage-displayed peptide libraries. The affinity selection process described thereafter enables a single researcher to efficiently probe the recognition profiles of numerous PRMs in a 1 week time period.
信号复合物通常涉及包含催化结构域和肽识别模块（PRM）的多结构域蛋白，这些模块介导蛋白质-蛋白质相互作用，并通过与含有核心序列基序的配体结合来组装复合物。与大规模物理相互作用筛选同时进行的是，人们投入了大量精力来阐明常见PRM的共有谱。我们在此描述了一种使用噬菌体展示肽库生成PRM共有谱的可靠且经过验证的方案。该方案的初始阶段需要将PRM作为融合蛋白进行克隆、表达和纯化，此外还要构建高度多样化的噬菌体展示肽库。此后描述的亲和选择过程使单个研究人员能够在1周时间内有效地探究众多PRM的识别谱。
