School of Chemistry, University of East Anglia, Norwich, UK.
Methods Mol Biol. 2023;2676:21-40. doi: 10.1007/978-1-0716-3251-2_2.
Genetically encoded site-specifically incorporated noncanonical amino acids (ncAAs) have been used to modulate properties of several proteins. Here, we describe a procedure for engineering photoactive antibody fragments that bind to their target antigen only after irradiation with 365 nm light. The procedure starts with identification of tyrosine residues in antibody fragments that are important for antibody-antigen binding and thus targets for replacement with photocaged tyrosine (pcY). This is followed by cloning of plasmids and expression of pcY-containing antibody fragments in E. coli. Finally, we describe a cost-effective and biologically-relevant method for measuring the binding affinity of photoactive antibody fragments to antigens expressed on the surface of live cancer cells.
基因编码的特异性整合非天然氨基酸（ncAAs）已被用于调节几种蛋白质的性质。在这里，我们描述了一种工程化光活性抗体片段的方法，这些抗体片段只有在 365nm 光照射后才能与靶抗原结合。该方法首先鉴定抗体片段中对抗体-抗原结合很重要的酪氨酸残基，这些残基是光笼酪氨酸（pcY）取代的目标。接下来是克隆质粒并在大肠杆菌中表达含有 pcY 的抗体片段。最后，我们描述了一种经济有效的、与生物相关的方法，用于测量光活性抗体片段与活癌细胞表面表达的抗原的结合亲和力。
