期刊
LIFE-BASEL
卷 13, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/life13101972
关键词
cell-free protein synthesis; thyroid receptor ligand; fusion protein; reporter enzyme
Thyroid receptor signaling plays a crucial role in physiological processes and its disruption can lead to severe disorders. Detection of thyroid receptor ligands is important for toxicology and pharmacology. A biosensor system was developed using cell-free protein synthesis and reengineered with different reporter systems, reducing the cost by 60%. This could potentially be a promising platform for high throughput screening and portable detection of human thyroid receptor ligands.
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 (hTR beta) receptor activator and a beta-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 (beta-gal), the alpha fragment of beta-gal (beta-gal-alpha), or a split alpha fragment of the beta-gal (split beta-gal-alpha). These biosensor constructs are deployed using E. coli XL1-Blue cell extract to (1) avoid the beta-gal background activity abundant in BL21 cell extract and (2) facilitate beta-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.
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