引入半胱氨酸实现纳米抗体的定点标记。

Introducing Cysteines into Nanobodies for Site-Specific Labeling.

机构信息

Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.

出版信息

Methods Mol Biol. 2022;2446:327-343. doi: 10.1007/978-1-0716-2075-5_16.

DOI:10.1007/978-1-0716-2075-5_16

Abstract

We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

摘要

我们开发了一种普遍适用于纳米抗体（Nb）框架区丝氨酸残基半胱氨酸突变的方法。该策略允许随后用硫醇反应性化合物标记，而不破坏 Nb 抗原结合。我们提供了一种用 Alexa Fluor 488-马来酰亚胺和汞化合物对氯汞苯甲酸（PCMB）生产、标记和测定半胱氨酸工程化 Nb（cys-Nb）的方案。Alexa Fluor 488 和 PCMB 标记的 cys-Nb 可分别用于免疫荧光显微镜和结晶学中的实验相测定。