R. Silvius, John Leventis, Rania A Novel “Prebinding” Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes We have examined quantitatively the efficiency and the kinetics of sortase A-mediated coupling of model substrate proteins (derived from green fluorescent protein and the SNAP variant of O-alkylguanine-DNA alkyltransferase) to large unilamellar liposomes incorporating low levels of oligopeptide-modified acceptor lipids. Under normal reaction conditions, even using high concentrations of S. aureus or S. pyogenes sortase A and optimal protein coupling substrates and acceptor lipids, protein–liposome coupling is slow, gives at best modest coupling yields, and is markedly limited by the hydrolytic activity of sortase. We demonstrate, however, that these limitations can be overcome under “prebinding” conditions that promote initial reversible association of sortase and the substrate protein with the liposome surface. Using oligohistidine-tagged sortase and substrate proteins and liposomes incorporating an acceptor lipid together with a Ni­(II)-chelating lipid derivative, high coupling rates and yields can be obtained at low sortase concentrations, while virtually eliminating adverse effects of sortase hydrolytic activity on protein coupling. The prebinding approach described here can readily be adapted, and if necessary rendered virtually “traceless”, to accommodate diverse protein coupling substrates and end uses of the protein-modified liposomes. substrate proteins;reaction conditions;liposome surface;oligohistidine-tagged sortase;unilamellar liposomes;SNAP variant;acceptor lipid;substrate protein;prebinding approach;acceptor lipids;pyogenes sortase;protein-modified liposomes;oligopeptide-modified acceptor lipids;hydrolytic activity;sortase hydrolytic activity;sortase A-mediated;O-alkylguanine-DNA alkyltransferase;sortase concentrations;model substrate proteins 2017-03-30
    https://acs.figshare.com/articles/journal_contribution/A_Novel_Prebinding_Strategy_Dramatically_Enhances_Sortase-Mediated_Coupling_of_Proteins_to_Liposomes/4828327
10.1021/acs.bioconjchem.7b00087.s001