Low-Cost Healthcare Diagnostics By Directed Evolution of Peptide Receptors in Yeast

Low cost, point-of-care (POC) diagnostics for specific proteins would have imminently useful applications in resource-poor healthcare, as well as counter-bioterrorism (detecting pathogens), and environmental monitoring (detecting contamination).  However, existing technologies for protein detection are either too expensive or too bulky for portability (mass spec) or not suitable for continuous monitoring and have limited analyte possibilities (lateral flow immunoassays, i.e., pregnancy tests).  To date, the majority of Synthetic Biology biosensing parts detect analytes in the cytoplasm and nucleus, which preclude most proteins that can not cross the membrane.

We have developed a directed evolution approach for tuning sensitivity and specificity of G-protein coupled receptors (GPCRs). Nature has already evolved mechanisms for sensitive and specific detection of peptides and proteins using GPCRs.  In particular the yeast GPCR, Ste2p, detects the 13 amino acid peptide called a-factor.  Evolving Ste2p to recognize different peptide sequences would be a relatively small evolutionary step.

Using a FACS-based high throughput screen, we have evolved receptors that can detect several different peptide sequences, including sequences with homology to important clinical biomarkers for renal failure and tuberculosis.  We have shown that we can alter the sensitivity and specificity of a receptor by selecting mutant receptors that respond to low concentrations of ligand (sensitivity) and not respond to non-target ligands until the concentration is very high (specificity).   Evolved receptors can discriminate peptides with only one amino acid variation.

This approach should have many useful applications in Synthetic Biology, including: healthcare diagnostics, novel cell-to-cell communication (as cells can be easily engineered to produces any number of peptide sequences), and to reveal sequence-activity relationships (SARs) for GPCRs, a class of drug-target receptors that have been difficult to characterize because of difficulties crystalizing transmembrane receptors.