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A Novel “Prebinding” Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes
journal contribution
posted on 2017-03-30, 00:00 authored by John R. Silvius, Rania LeventisWe 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.
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substrate proteinsreaction conditionsliposome surfaceoligohistidine-tagged sortaseunilamellar liposomesSNAP variantacceptor lipidsubstrate proteinprebinding approachacceptor lipidspyogenes sortaseprotein-modified liposomesoligopeptide-modified acceptor lipidshydrolytic activitysortase hydrolytic activitysortase A-mediatedO-alkylguanine-DNA alkyltransferasesortase concentrationsmodel substrate proteins
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