David H. Koch Center, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Nat Protoc. 2013 Oct;8(10):1916-39. doi: 10.1038/nprot.2013.119. Epub 2013 Sep 12.
Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ∼8 weeks.
技术主要用于蛋白质相互作用的研究和细胞内受体的发现，如亲和捕获复合物纯化和酵母双杂交系统，由于蛋白质的不溶性、瞬时或弱的蛋白质相互作用或不相关的细胞内环境，可能会产生不准确的数据集。噬菌体展示技术是克服这些限制的一种通用工具，也可用于潜在地创造疫苗和工程肽和抗体作为靶向诊断和治疗剂。我们最近开发了一种新的技术，用于筛选内化噬菌体（iPhage）载体和文库，使用配体/受体非依赖性机制穿透真核细胞。iPhage 颗粒为细胞器配体及其相应受体的组合细胞内靶向以及活细胞中功能蛋白结构域的指纹鉴定提供了独特的发现平台。在这里，我们解释了基于 iPhage 的载体和文库的设计、克隆、构建和生产，以及细胞器受体的基本配体-受体鉴定和验证方法学。iPhage 文库筛选可以在 8 周内完成。
