UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California.
Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California.
Curr Protoc. 2024 Jan;4(1):e960. doi: 10.1002/cpz1.960.
Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.
蛋白质展示系统是一种强大的技术，用于鉴定与目标蛋白具有高亲和力结合的蛋白分子。实施展示系统的最初挑战是构建高多样性的原始文库。在这里，我们描述了使用简并寡核苷酸生成设计的锚蛋白重复蛋白（DARPin）展示文库的方法。具体描述了通过重叠延伸 PCR 构建单个 DARPin 重复模块、通过限制性内切酶消化和连接将模块串联、以及将串联的模块并入含有亲水 N 和 C 末端盖帽结构域的全长 DARPin 序列的细菌克隆或展示载体中。包括用于通过下一代测序估计原始和富集文库多样性的 DARPin 序列 PCR 扩增的方案，以及一个用于分析原始和富集序列的简单基于 Linux 的程序。© 2024 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：通过重叠延伸 PCR 生成单个 DARPin 重复基本方案 2：DARPin 重复的串联基本方案 3：将内部重复连接到含有 N 和 C 末端盖帽重复的克隆/展示载体中基本方案 4：通过下一代测序（NGS）估计文库大小和多样性基本方案 5：原始和富集文库的 NGS 分析。
