CF2H: a Cell-Free Two-Hybrid platform for rapid protein binder screening
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Abstract
Protein binders that detect, activate, inhibit or otherwise modulate their targets are pivotal for biomedical applications. With the increasing accuracy and accessibility of de novo protein design, faster and cheaper experimental screening methods would democratize and accelerate the identification of high-affinity binders. Here we present Cell-Free Two-Hybrid (CF2H), a rapid and sensitive method for detecting high-affinity protein-protein interactions (PPI) that does not require cloning, protein purification nor high-end laboratory equipment. CF2H uses a dimerization-activated DNA binding domain (DBD) fused to prey and bait proteins to trigger transcription upon protein-protein interaction. We demonstrate that CF2H enables the detection of interactions between various types of target and binder proteins such as single-domain antibodies, DARPins and de novo designed binders. We benchmark CF2H as a screening platform by validating previously reported binders for Mdm2 and discover high-affinity binders targeting the checkpoint inhibitor PD-L1 in less than 24 hours. Finally, we show that CF2H can be used to characterize small-molecules modulators of PPI and detect protein biomarkers, opening the door for a new class of cell-free biosensors.
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Here we developed CF2H, a rapid and simple workflow for high-affinity binder screening. Our system was designed with the aim of making binder screening simpler, more affordable and accessible to the community. By combining an E. coli lysate-based cell-free system compatible with expression from linear DNA templates to a two-hybrid approach, we constructed a workflow that bypasses tedious cloning, culturing and sequencing steps. Our experimental setup only requires a set of pipettes and a microplate reader for GFP measurements, and its complexity is comparable to setting up PCR reactions.
This is a really interesting method and was a super fun read! I like that not only can it clearly accelerate binder studies, but it can be done in any lab. I'm looking for a use case to try it out already!
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In addition to binding affinity, differences in expression levels, solubility, and stability among binders likely influence the signal output.
Can you interrogate the effect of these different features on the signal output in this method?
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