Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors.
J Porter HuntTyler J FreeJackelyn GaliardiKevin M WattDavid W WoodBradley C BundyPublished in: Life (Basel, Switzerland) (2023)
Thyroid receptor signaling controls major physiological processes and disrupted signaling can cause severe disorders that negatively impact human life. Consequently, methods to detect thyroid receptor ligands are of great toxicologic and pharmacologic importance. Previously, we reported thyroid receptor ligand detection with cell-free protein synthesis of a chimeric fusion protein composed of the human thyroid receptor beta (hTRβ) receptor activator and a β-lactamase reporter. Here, we report a 60% reduction in sensing cost by reengineering the chimeric fusion protein biosensor to include a reporter system composed of either the full-length beta galactosidase (β-gal), the alpha fragment of β-gal (β-gal-α), or a split alpha fragment of the β-gal (split β-gal-α). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the β-gal background activity abundant in BL21 cell extract and (2) facilitate β-gal complementation reporter activity to detect human thyroid receptor ligands. These results constitute a promising platform for high throughput screening and potentially the portable detection of human thyroid receptor ligands.
Keyphrases
- endothelial cells
- cell free
- cell therapy
- induced pluripotent stem cells
- escherichia coli
- label free
- gold nanoparticles
- crispr cas
- oxidative stress
- single cell
- stem cells
- immune response
- binding protein
- early onset
- high throughput
- quantum dots
- sensitive detection
- bone marrow
- multidrug resistant
- toll like receptor
- loop mediated isothermal amplification
- circulating tumor