Cell-free production of fluorescent proteins for the discovery of novel ribosome-targeting antibiotics.

Various issues including the overuse of antibiotics has led to the development of threatening multidrug-resistant bacterial strains urging development of novel anti-infectives. One quarter of current clinical phase III antibiotic drug candidates address ribosomal protein translation as a target. Here, we describe an effective cell-free in vitro screening system for inhibitors of bacterial ribosome activity with direct fluorescence read-out. Using ribosomal S30 extracts from Escherichia coli, Salmonella enterica, and Pseudomonas putida, the validity of this system is demonstrated by concentration-dependent inhibition of translation by a set of different classes of translation-targeting drugs. The single-compartment cell-free translation reaction is compatible with multi-well formats. Fluorophore formation of green fluorescent protein or monomeric NeonGreen occurs in an hour time frame without the need of adding reagents for secondary enzymatic detection saving handling time, and prohibiting false positives. As label-free readout, the dose response further allows for IC

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.