Knob-in-hole antibodies can be utilized to introduce a single tag for chemo-enzymatic functionalization. By either introducing a single C-terminal sortase tag (sortase-tag expressed protein ligation) or tyrosine tag (G4Y), mono-functionalization of the monoclonal antibody trastuzumab was achieved rapidly and in high yields. This method was applied to selectively and efficiently introduce a single fluorescent tag, cytokine or single-chain variable fragment, as well as produce clean homo dimers of trastuzumab.
Bruins, J. J., van de Wouw, C., Koen, W., Lina, B., Albada, B., & van Delft, F. L. (2019). Highly efficient mono-functionalization of knob-in-hole antibodies with strain-promoted click chemistry. Journal of the American Chemical Society, 4, 11801-11807. . https://doi.org/10.1021/acsomega.9b01727