Engineering Homogeneous Photoactive Antibody Fragments

Thomas Bridge, Amit Sachdeva

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review


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.
Original languageEnglish
Title of host publicationGenetically Incorporated Non-Canonical Amino Acids
EditorsYu-Hsuan Tsai, Simon J. Elsässer
Number of pages20
ISBN (Electronic)978-1-0716-3251-2
ISBN (Print)978-1-0716-3250-5
Publication statusPublished - 6 Jun 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Antibodies
  • Cancer
  • Genetic code expansion
  • Light-activated biotherapeutics
  • Noncanonical amino acids
  • Photocaged amino acids
  • Synthetic biology

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