Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox

Irsyad N. A. Khairil Anuar, Anusuya Banerjee, Anthony H. Keeble, Alberto Carella, Georgi I. Nikov and Mark Howarth ()
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Irsyad N. A. Khairil Anuar: University of Oxford, South Parks Road
Anusuya Banerjee: University of Oxford, South Parks Road
Anthony H. Keeble: University of Oxford, South Parks Road
Alberto Carella: University of Oxford, South Parks Road
Georgi I. Nikov: University of Oxford, South Parks Road
Mark Howarth: University of Oxford, South Parks Road

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: Abstract Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy&Go enabled sensitive purification of SpyTag-fusions from Escherichia coli, giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy&Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy&Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling.

Date: 2019
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DOI: 10.1038/s41467-019-09678-w

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