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, Mark Howarth

Research output: Contribution to journalArticleScientificpeer-review

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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.

Original languageEnglish
Article number3714
JournalNature Communications
Issue number1
Publication statusPublished - Dec 2022
Publication typeA1 Journal article-refereed

Publication forum classification

  • Publication forum level 3

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • General
  • Physics and Astronomy(all)


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