用于细胞蛋白VIPER标记的CoilR探针肽的生成。
Generation of CoilR Probe Peptides for VIPER-labeling of Cellular Proteins.
作者信息
Doh Julia K, Tobin Savannah J, Beatty Kimberly E
机构信息
Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon 97239, USA.
OHSU Center for Spatial Systems Biomedicine, Oregon Health & Science University, Portland, Oregon 97239, USA.
出版信息
Bio Protoc. 2019 Nov 5;9(21):e3412. doi: 10.21769/BioProtoc.3412.
Versatile Interacting Peptide (VIP) tags are a new class of genetically-encoded tag designed for imaging cellular proteins by fluorescence and electron microscopy. In 2018, we reported the VIPER tag ( Doh , 2018 ), which contains two elements: a genetically-encoded peptide tag (, CoilE) and a probe peptide (, CoilR). These two peptides deliver contrast to a protein of interest by forming a specific, high-affinity heterodimer. The probe peptide was designed with a single cysteine residue for site-specific modification via thiol-maleimide chemistry. This feature can be used to attach a variety of biophysical reporters to the peptide, including bright fluorophores for fluorescence microscopy or electron-dense nanoparticles for electron microscopy. In this Bio-Protocol, we describe our methods for expressing and purifying recombinant CoilR. Additionally, we describe protocols for making fluorescent or biotinylated probe peptides for labeling CoilE-tagged cellular proteins. This protocol is complemented by two other Bio-Protocols outlining the use of VIPER ( Doh , 2019a and 2019b).
多功能相互作用肽（VIP）标签是一类新的基因编码标签，旨在通过荧光和电子显微镜对细胞蛋白质进行成像。2018年，我们报道了VIPER标签（Doh，2018），它包含两个元件：一个基因编码肽标签（CoilE）和一个探针肽（CoilR）。这两种肽通过形成特定的、高亲和力的异二聚体为感兴趣的蛋白质提供对比度。探针肽设计有一个半胱氨酸残基，用于通过硫醇-马来酰亚胺化学进行位点特异性修饰。这一特性可用于将各种生物物理报告分子连接到该肽上，包括用于荧光显微镜的明亮荧光团或用于电子显微镜的电子致密纳米颗粒。在本生物协议中，我们描述了表达和纯化重组CoilR的方法。此外，我们还描述了制备用于标记CoilE标记的细胞蛋白的荧光或生物素化探针肽的方案。本方案由另外两个概述VIPER使用方法的生物协议（Doh，2019a和2019b）补充。
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