DeVisch, A. | bioRxiv Science

bioRxiv2025

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

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.

synthetic biology↗

bioRxiv2021

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from {sigma}70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the {Delta}recBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

synthetic biology↗