N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone

Brune, Karl D; Lieknina, Ilva; Sutov, Grigorij; Morris, Alexander R; Jovicevic, Dejana; Kalnins, Gints; Kazaks, Andris; Kluga, Rihards; Kastaljana, Sabine; Zajakina, Anna; Jansons, Juris; Skrastina, Dace; Spunde, Karina; Cohen, Alexander A; Bjorkman, Pamela J; Morris, Howard R; Suna, Edgars; Tars, Kaspars

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in†vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH†7.5 in 1†h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.