“Fluorescent minihelix assay for translation quantification in high-throughput cell-free systems”

We report the use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter in vitro with the Flash dye to study cell-free protein expression dynamics and engineered ribosome activity. The fluorescent product can be detected and quantified via its characteristic emission spectrum in a standard 96/384-well plate reader, RT-qPCR system, or gel electrophoresis. The fluorescent reporter helix is short enough to be encoded on a primer pair and can tag any protein of interest via PCR. Both tagged protein or standalone reporter can be detected in real time during and in terminal cell-free expression reactions, or in gel, without the need for staining. The fluorescent signal is stable and linearly correlates with protein concentration, thus is suitable for product quantification. Finally, we demonstrate how this reporter can be used for future efforts in engineering in vitro translation systems.