School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Maurice Wilkins Centre, University of Auckland, Auckland, New Zealand.
Methods Mol Biol. 2020;2136:377-395. doi: 10.1007/978-1-0716-0467-0_30.
Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.
分子内异肽键是由赖氨酸（Lys）和天冬氨酸/天冬酰胺（Asn/Asp）侧链之间自动催化形成的，广泛存在于革兰氏阳性菌黏附素的免疫球蛋白样结构域中，包括 A 组链球菌，赋予其相当大的机械和化学稳定性。这些特性使它们成为生物技术应用中的理想选择。本文详细介绍了在 Ig 样蛋白中构建异肽键所涉及的实际考虑因素，包括选择可以引入键形成残基的位点以及合适的诱变方法。我们指定了如何确定是否形成了异肽键，可以采用哪些策略来克服问题，以及如何监测工程蛋白的稳定性。
