SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly

Susan K. Vester, Rolle Rahikainen, Irsyad N. A. Khairil Anuar, Rory A. Hills, Tiong Kit Tan and Mark Howarth ()
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Susan K. Vester: University of Oxford
Rolle Rahikainen: University of Oxford
Irsyad N. A. Khairil Anuar: University of Oxford
Rory A. Hills: University of Oxford
Tiong Kit Tan: University of Oxford
Mark Howarth: University of Oxford

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: Abstract Proteins can be empowered via SpyTag for anchoring and nanoassembly, through covalent bonding to SpyCatcher partners. Here we generate a switchable version of SpyCatcher, allowing gentle purification of SpyTagged proteins. We introduce numerous histidines adjacent to SpyTag’s binding site, giving moderate pH-dependent release. After phage-based selection, our final SpySwitch allows purification of SpyTag- and SpyTag003-fusions from bacterial or mammalian culture by capture at neutral pH and release at pH 5, with purity far beyond His-tag methods. SpySwitch is also thermosensitive, capturing at 4 °C and releasing at 37 °C. With flexible choice of eluent, SpySwitch-purified proteins can directly assemble onto multimeric scaffolds. 60-mer multimerization enhances immunogenicity and we use SpySwitch to purify receptor-binding domains from SARS-CoV-2 and 11 other sarbecoviruses. For these receptor-binding domains we determine thermal resilience (for mosaic vaccine development) and cross-recognition by antibodies. Antibody EY6A reacts across all tested sarbecoviruses, towards potential application against new coronavirus pandemic threats.

Date: 2022
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DOI: 10.1038/s41467-022-31193-8

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