Antimicrobial Resistance, Omics and Microbiota Group, Department of Biosciences, Nottingham Trent University, Nottingham, UK.
Section for Evolution and Genetics, Department of Biosciences, University of Oslo, Oslo, Norway.
Methods Mol Biol. 2024;2778:53-63. doi: 10.1007/978-1-0716-3734-0_4.
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.
SpyCatcher-SpyTag 系统已成为蛋白质连接的一种流行且多功能的工具。它基于一个小的球形蛋白（SpyCatcher），该蛋白与一个 13 个残基的肽（SpyTag）结合，随后导致形成共价异肽键。因此，该反应基本上是不可逆的。在这里，我们描述了如何使用 SpyCatcher-SpyTag 系统来标记表面暴露的细菌外膜蛋白，例如，用于拓扑作图或荧光时程实验。在本章中，我们介绍了如何使用荧光测量和显微镜来测量与超折叠 GFP 融合的 SpyCatcher 的标记效率。
