Covalent Attachment of BG-Linker-AURIF

Monomethyl auristatin F (MMAF) was sourced from BrightGene Bio-Medical Technology (China) and the compound BG-modified to generate BG–linker–AURIF by Professor Roger Hunter’s organic synthesis group, Department of Chemistry, University of Cape Town, South Africa. Detailed information about the synthesis, spectroscopic, and analytical data for the BG–linker–AURIF product is provided in Huysamen et al. (2023 (link)) (supplementary information). The purified recombinant fusion protein was incubated for 4 h at room temperature, with a threefold molar excess of BG–linker–AURIF [initially in lyophilized form, BG–linker–AURIF was solubilized in 100% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich, South Africa), 1 M ectoine (a protein-stabilizing compatible solute (Lippert and Galinski 1992 ) (Sigma-Aldrich, South Africa) in 1 × PBS and 1 mM DTT]. The unconjugated BG–linker–AURIF was removed using 10 K-sized Amicon filters (Sigma-Aldrich, South Africa) according to the manufacturer’s instructions. Since the resulting product cannot be directly visualized, saturation of the binding domain of SNAP-tag with BG–linker–AURIF was ascertained by post-incubation (double conjugation) with a twofold molar excess of BG-Alexa Fluor 488 for 1 h at 37 °C. Next, SDS-PAGE analysis was conducted, and visualization of any potential fluorescence signal was carried out as described previously.