Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 141, Issue 20, Pages 8128-8135Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.8b12298
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Funding
- Australian Research Council [DP160100973, DP150100936, IC160100027]
- NHMRC [APP1113262]
- CSIRO Synthetic Biology Future Science Fellowship from the CSIRO Synthetic Biology Future Science Platform
- ITTC [IC160100027]
- QUT/CSIRO Synthetic Biology alliance
- Molecular Warehouse Inc.
- Australian Research Council [IC160100027] Funding Source: Australian Research Council
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Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.
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