Generalizable Protein Biosensors Based on Synthetic Switch Modules
| Autor: | Wayne A. Johnston, Selvakumar Edwardraja, Zhenling Cui, Jason Whitfield, Fernanda Ely, Elvira Wijker, Ignacio Retamal Lantadilla, Jacobus P.J. Ungerer, Patricia M. Walden, Claudia E. Vickers, Zhong Guo, Kirill Alexandrov |
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| Rok vydání: | 2019 |
| Předmět: |
Calmodulin
Recombinant Fusion Proteins Glucose Dehydrogenases Allosteric regulation Serum Albumin Human Biosensing Techniques Computational biology Protein Engineering 010402 general chemistry 01 natural sciences Biochemistry Tacrolimus Catalysis Tacrolimus Binding Proteins 03 medical and health sciences Colloid and Surface Chemistry Bacterial Proteins Glucose dehydrogenase Diabetes Mellitus Humans Amino Acid Sequence Acinetobacter calcoaceticus Saliva 030304 developmental biology 0303 health sciences biology Chemistry Activator (genetics) General Chemistry Protein engineering Biochemical Activity Ligand (biochemistry) 0104 chemical sciences Cyclosporine biology.protein Calmodulin-Binding Proteins alpha-Amylases Biosensor Biomarkers |
| Zdroj: | Journal of the American Chemical Society. 141:8128-8135 |
| ISSN: | 1520-5126 0002-7863 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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