Alkenyl-sfGFP is fluorescently labeled with dansyl-thiol, and bioconjugated to lysozyme to assemble a non-linear protein dimer via the thiol-ene reaction.
(A) sfGFP bearing an alkene functionality reacts photochemically with dansyl-thiol (10) or lysozyme (LYZ). (B) SDS-PAGE analysis demonstrates the labeling of alkenyl-sfGFP with 10 after 5 min of UV irradiation via thiol-ene ligation (lanes 5 and 6). Fluorescence (top) and Coomassie stain (bottom). (C) SDS-PAGE analysis shows mobility band shifts from 28 kD to 44 kD after samples were UV irradiated for 10 min (lanes 8 and 9), corresponding to the molecular weight of sfGFP-lysozyme conjugate. WT: wild-type sfGFP; 1 and 2: sfGFP carrying the corresponding UAA; LYZ: lysozyme. –UV: samples were not exposed to UV irradiation. +UV: samples were irradiated at 365 nm for 5 or 10 min.