A protein switch couples an input signal (e.g. ligand concentration) to an output response (e.g. enzyme activity). Applications for protein switches include use as genetically encoded biosensors, protein therapeutics and building blocks to create novel signaling or regulatory networks. We have previously engineered a family of protein switches by the in-vitro recombination of two non-homologous genes encoding maltose binding protein (MBP) and TEM1 beta-lactamase (BLA). These switches exhibited maltose-dependent BLA activity. To aid in the development of new protein switches, we engineered an E. coli strain to behave as a band-pass filter for enzyme activity; thus, only cells with enzyme activities within a specific, narrow range will grow. The key feature of this system is the ability to externally tune the band's location within a four order of magnitude range by the addition of a compound to the growth media. Using this system, we isolated several MBP-BLA protein switches from a library containing inactive and non-switching members. From this library we also isolated several non-switching members that show ligand-stabilized expression; members that were not discovered using a traditional selection. Finally, this selection system was applied to a computationally-designed library in which we have attempted to convert our maltose-activated switch into one that is specifically activated by sucrose.

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