VHHs fused to the natural llama heavy-chain antibody long hinge region, and a hexahistidine (his6) tag was produced in baker’s yeast using vector pRL188 and purified from culture supernatant using immobilized-metal affinity chromatography as described earlier [23 (link)]. VHHs produced in this manner were indicated by the suffix “F.” About 10% of the VHH amount produced in this manner was dimerized through the single cysteine present at the C-terminus of the VHH, immediately preceding the his6 tag. Both monomeric and dimeric VHHs are useful for functional immobilization of VHHs to polystyrene surfaces by passive adsorption [9 (link),24 (link)]. Yeast-produced VHHs were biotinylated at a weight ratio of protein to biotin of 5 using amine-reactive sulfo-N-hydroxysuccinimide-LC-biotin (ThermoFisher Scientific).
VHHs were subjected to reducing SDS-PAGE using precast gels (Novex, San Diego, CA, USA), and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions.
VHHs were subjected to reducing SDS-PAGE using precast gels (Novex, San Diego, CA, USA), and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions.
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