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

Karl D. Brune, Ilva Liekniņa, Grigorij Sutov, Alexander R. Morris, Dejana Jovicevic, Gints Kalniņš, Andris Kazāks, Rihards Kluga, Sabine Kastaljana, Anna Zajakina, Juris Jansons, Dace Skrastiņa, Karīna Spunde, Alexander A. Cohen, Pamela J. Bjorkman, Howard R. Morris, Edgars Suna, Kaspars Tārs


Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a nonenzymatic, post-translational modification (PTM) of N-terminal HisTags. In this publication, BioPharmaSpec scientists collaborate with scientists from Genie Biotech, a biotechnology company specialized in bioconjugation, to report high-yield, site-selective in vitro α-aminoacylation of peptidesglycoproteinsantibodies, 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, azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) is multimerized 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. The paper details 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.

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