Engineering Homogeneous Photoactive Antibody Fragments

Bridge, Thomas and Sachdeva, Amit (2023) Engineering Homogeneous Photoactive Antibody Fragments. In: Genetically Incorporated Non-Canonical Amino Acids. Methods in Molecular Biology . Springer, pp. 21-40. ISBN 978-1-0716-3250-5

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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.

Item Type: Book Section
Uncontrolled Keywords: antibodies,cancer,genetic code expansion,light-activated biotherapeutics,noncanonical amino acids,photocaged amino acids,synthetic biology,molecular biology,genetics ,/dk/atira/pure/subjectarea/asjc/1300/1312
Faculty \ School: Faculty of Science > School of Chemistry
UEA Research Groups: Faculty of Science > Research Groups > Chemistry of Life Processes
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Depositing User: LivePure Connector
Date Deposited: 21 Aug 2023 09:30
Last Modified: 25 Sep 2023 08:31
DOI: 10.1007/978-1-0716-3251-2_2

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