title: A Novel "Prebinding" Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes.
abstract: 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.
authors.affiliation: Department of Biochemistry, McGill University , 3655 Promenade Sir-William-Osler, Montréal, QC, Canada H3G 1A9.
authors.forename: John R
authors.identifier: 0000-0003-4050-509X
authors.initials: JR
authors.lastname: Silvius
authors.affiliation: Department of Biochemistry, McGill University , 3655 Promenade Sir-William-Osler, Montréal, QC, Canada H3G 1A9.
authors.forename: Rania
authors.initials: R
authors.lastname: Leventis
mesh_terms: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D011485:Protein Binding; D013297:Streptococcus pyogenes
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases