CF2H: a Cell-Free Two-Hybrid platform for rapid protein binder screening

Nature Communications (Mar 2026) CF2H: a cell-free two-hybrid platform for rapid protein binder screening Abstract 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 discovering 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-molecule modulators of PPI and detect protein biomarkers, opening the door for a new class of cell-free biosensors.