Nanobodies are single-domain antibody fragments that have found widespread use in basic research, therapy, and diagnostics. Like other antibody formats, nanobodies can be developed with high affinity and specificity for desired antigens. A photobody is a light-activatable nanobody, obtained by incorporating a photo-labile caging group into the paratope region. The caging group prevents antigen binding until removed with light (365 nm), thereby rendering the binding controllable with high temporal and spatial resolution. Thus far photocaged tyrosine residues have been used for this purpose, as tyrosine is a frequent residue at critical positions of nanobody paratopes. Nanobodies without a tyrosine residue at the antigen-binding interface may require a different strategy. In this chapter, we describe methods to design and prepare photobodies by recombinant expression in Escherichia coli in combination with genetic code expansion technology to incorporate ortho-nitrobenzyl-tyrosine residues. We use the conversion of the anti-green fluorescent protein enhancer nanobody into a photobody as an example. These protocols should be applicable to many other nanobodies.
Keywords: Cage group; Genetic code expansion; Nanobody; O-nitrobenzyl-tyrosine; Photobody; Photocaging; VHH.
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