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Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments
Author(s) -
Li Zeyang,
Theile Christopher S.,
Chen GuanYu,
Bilate Angelina M.,
Duarte Joao N.,
Avalos Ana M.,
Fang Tao,
Barberena Roberto,
Sato Shuji,
Ploegh Hidde L.
Publication year - 2015
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201505277
Subject(s) - holliday junction , fluorophore , fluorescence , chemistry , biophysics , conjugated system , sortase , biochemistry , biology , polymer , dna , optics , physics , homologous recombination , organic chemistry , bacterial protein , gene
The site‐specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self‐quenching from 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. These fluorescent Holliday junctions improve fluorescence yields for both single‐domain and full‐sized antibodies without deleterious effects on antigen binding.