Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Muenster, Münster, Germany.
Methods Mol Biol. 2022;2446:409-424. doi: 10.1007/978-1-0716-2075-5_21.
Nanobodies are single-domain antibody fragments that have found widespread use in basic research, therapy, and diagnostics. Like other antibody formats, nanobodies can be developed with high affinity and specificity for desired antigens. A photobody is a light-activatable nanobody, obtained by incorporating a photo-labile caging group into the paratope region. The caging group prevents antigen binding until removed with light (365 nm), thereby rendering the binding controllable with high temporal and spatial resolution. Thus far photocaged tyrosine residues have been used for this purpose, as tyrosine is a frequent residue at critical positions of nanobody paratopes. Nanobodies without a tyrosine residue at the antigen-binding interface may require a different strategy. In this chapter, we describe methods to design and prepare photobodies by recombinant expression in Escherichia coli in combination with genetic code expansion technology to incorporate ortho-nitrobenzyl-tyrosine residues. We use the conversion of the anti-green fluorescent protein enhancer nanobody into a photobody as an example. These protocols should be applicable to many other nanobodies.
纳米抗体是单域抗体片段，已广泛应用于基础研究、治疗和诊断。与其他抗体形式一样，纳米抗体可以针对所需抗原进行高亲和力和特异性的开发。光抗体是一种光活化的纳米抗体，通过将光不稳定的笼状基团引入抗原结合部位获得。该笼状基团可防止抗原结合，直到用光（365nm）去除，从而使结合具有高时间和空间分辨率的可控性。到目前为止，已经使用光笼酪氨酸残基来实现这一目的，因为酪氨酸是纳米抗体抗原结合部位关键位置的常见残基。在抗原结合界面处没有酪氨酸残基的纳米抗体可能需要不同的策略。在本章中，我们描述了通过在大肠杆菌中的重组表达并结合遗传密码扩展技术来设计和制备光抗体的方法，以掺入邻硝基苄基酪氨酸残基。我们以将抗绿色荧光蛋白增强子纳米抗体转化为光抗体为例。这些方案应该适用于许多其他的纳米抗体。
