使用 SpyCatcher-SpyTag 系统对外膜蛋白质进行荧光标记。

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2024-01-01 DOI:10.1007/978-1-0716-3734-0_4
Rachael Duodu, Dirk Linke, Jack C Leo
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引用次数: 0

摘要

SpyCatcher-SpyTag 系统已成为蛋白质连接方面一种流行的多功能工具。该系统基于一种小球状蛋白质(SpyCatcher)与 13 个残基的多肽(SpyTag)结合,随后形成共价异肽键。因此,该反应基本上是不可逆的。在此,我们将介绍如何利用 SpyCatcher-SpyTag 系统标记表面暴露的细菌外膜蛋白,例如用于拓扑图绘制或荧光时间历程实验。在本章中,我们将介绍如何利用荧光测量和显微镜来测量 SpyCatcher 与超夹层 GFP 融合后的标记效率。
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Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System.

The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcher fused with superfolder GFP in this chapter.

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来源期刊
Methods in molecular biology
Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
2.00
自引率
0.00%
发文量
3536
期刊介绍: For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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