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

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.


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Software and code
Policy information about availability of computer code Data collection SDS-PAGE images were collected by Image Lab 6.0.1. ITC data were collected by MicroCal PEAQ-ITC Analysis software version 1.1.0.1262. DLS data were collected by OmniSIZE 3.0. SEC-MALS data were collected by Astra 6.1. Cell data were collected by SoftMax Pro 5.4. Resin coupling efficiency data were collected by NanoDrop 1000 3.8.1. SpyTag-mClover3 fluorescence data were collected by CLARIOstar version 5.20 RS. Mass spectrometry data were collected by Mass Hunter Qualitative Analysis software version 7.0. Data analysis SDS-PAGE images were collected by Image Lab 6.0.1. PDB files were accessed by PyMOL 2.3. ITC data were analyzed and plotted in MicroCal PEAQ-ITC Analysis software version 1.1.0.1262. DLS data were analyzed by OmniSIZE 3.0 and plotted using Microsoft Excel 365 version 16.16.7. SEC-MALS data were analyzed by Astra 6.1 and plotted using GraphPad Prism version 7.0. Cell data were analyzed by OriginPro 2015. Coupling efficiency data were analyzed by Microsoft Excel 365 version 16.16.7. SpyTag-mClover3 fluorescence data were analyzed by Microsoft Excel 365 version 16.16.7. Mass spectrometry data were analyzed by Mass Hunter Qualitative Analysis software version 7.0 and plotted in OriginPro 2015. Protein sequences were analyzed by ExPASy ProtParam.
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