Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments.
Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments.
复制标题
DOI：
10.1002/anie.201505277
复制
发表时间：
2015-09-28
期刊：
影响因子：
--
通讯作者：
Ploegh HL
中科院分区：
文献类型：
--
作者：
Li Z;Theile CS;Chen GY;Bilate AM;Duarte JN;Avalos AM;Fang T;Barberena R;Sato S;Ploegh HL
用荧光团对蛋白质进行定点修饰可以在不影响其功能的情况下使蛋白质产生荧光。为了避免安装在附近的多个荧光团的自我猝灭，我们使用Holliday连接标记蛋白质。Holliday结能够以合理精确的间距使用多个荧光团进行修饰。我们设计了一个Holliday接头，它的四个臂中的三个用所选的荧光团修饰，其余的臂配备了二苯并环辛基取代基，使其与叠氮修饰的荧光单域抗体片段或完整的免疫球蛋白发生反应，这些抗体片段或完整的免疫球蛋白是在索尔特酶催化的反应中产生的。我们得出结论，荧光Holliday连接提高了单域抗体和全尺寸抗体的荧光产量，而不会对抗原结合产生有害影响。
Site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching of multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified, fluorescent single domain antibody fragment or an intact immunoglobulin, produced in a sortase-catalyzed reaction. We conclude that fluorescent Holliday junctions improve fluorescence yields for both single domain and full-sized antibodies, without deleterious effects on antigen binding.