TY - JOUR
T1 - SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly
AU - Vester, Susan K.
AU - Rahikainen, Rolle
AU - Khairil Anuar, Irsyad N.A.
AU - Hills, Rory A.
AU - Tan, Tiong Kit
AU - Howarth, Mark
N1 - Funding Information:
S.K.V., R.R. and M.H. were funded by the Biotechnology and Biological Sciences Research Council (BBSRC grant BB/T004983/1, to M.H.). I.N.A.K.A. was funded by Yayasan Khazanah, Oxford Centre for Islamic Studies, and St. John’s College Oxford. R.A.H. was funded by the Rhodes Trust. T.K.T. was funded by the EPA Cephalosporin Early Career Teaching and Research Fellowship and Townsend-Jeantet Prize Charitable Trust (Charity Number 1011770). M.H. and T.K.T. also thank the University of Oxford COVID-19 Research Response Fund and its donors (reference 0009517). We thank Niels Wicke and Dr. Anthony Keeble (both Howarth group) for helpful discussions on selections and biophysical analysis. Dr. Alexander Cohen and Prof. Pamela Bjorkman (both Caltech) kindly provided the RBD-SpyTag003 plasmids. We thank Prof. Alain Townsend (University of Oxford) for helpful discussions on sarbecovirus immune responses. We are grateful to Dr. David Staunton of the University of Oxford Department of Biochemistry Biophysical Suite for assistance. We thank Dr. Anthony Tumber of the University of Oxford Department of Chemistry for assistance with MS, supported by the BBSRC (BB/R000344/1). This research was funded in whole or in part by BBSRC grant BB/T004983/1. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.
Funding Information:
S.K.V., R.R. and M.H. were funded by the Biotechnology and Biological Sciences Research Council (BBSRC grant BB/T004983/1, to M.H.). I.N.A.K.A. was funded by Yayasan Khazanah, Oxford Centre for Islamic Studies, and St. John’s College Oxford. R.A.H. was funded by the Rhodes Trust. T.K.T. was funded by the EPA Cephalosporin Early Career Teaching and Research Fellowship and Townsend-Jeantet Prize Charitable Trust (Charity Number 1011770). M.H. and T.K.T. also thank the University of Oxford COVID-19 Research Response Fund and its donors (reference 0009517). We thank Niels Wicke and Dr. Anthony Keeble (both Howarth group) for helpful discussions on selections and biophysical analysis. Dr. Alexander Cohen and Prof. Pamela Bjorkman (both Caltech) kindly provided the RBD-SpyTag003 plasmids. We thank Prof. Alain Townsend (University of Oxford) for helpful discussions on sarbecovirus immune responses. We are grateful to Dr. David Staunton of the University of Oxford Department of Biochemistry Biophysical Suite for assistance. We thank Dr. Anthony Tumber of the University of Oxford Department of Chemistry for assistance with MS, supported by the BBSRC (BB/R000344/1). This research was funded in whole or in part by BBSRC grant BB/T004983/1. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
U2 - 10.1038/s41467-022-31193-8
DO - 10.1038/s41467-022-31193-8
M3 - Article
C2 - 35764623
AN - SCOPUS:85133025298
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 3714
ER -
