Keywords

cell-free protein synthesis, thyroid receptor ligand, fusion protein, reporter enzyme

Abstract

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.

Original Publication Citation

Hunt, J. P., Free, T. J., Galiardi, J., Watt, K. M., Wood, D. W., & Bundy, B. C. (2023). Streamlining the Detection of Human Thyroid Receptor Ligand Interactions with XL1-Blue Cell-Free Protein Synthesis and Beta-Galactosidase Fusion Protein Biosensors. Life, 13(10), 1972. https://doi.org/10.3390/life13101972

Document Type

Peer-Reviewed Article

Publication Date

2023-09-27

Publisher

Life

Language

English

College

Ira A. Fulton College of Engineering

Department

Chemical Engineering

University Standing at Time of Publication

Full Professor

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