title: Development of a synthetic receptor protein for sensing inflammatory mediators interferon-γ and tumor necrosis factor-α.
abstract: Intestinal inflammation has been implicated in a number of diseases, including diabetes, Crohn's disease, and irritable bowel syndrome. Important components of inflammation are interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are elevated both on the luminal and submucosal sides of the intestinal epithelial barrier in several diseases. Here, we developed a novel Escherichia coli based detection system for IFN-γ and TNF-α comprised of a chimeric protein and a simple signal transduction construct, which could be deployed on the luminal side of the intestine. OmpA of E. coli was engineered to detect IFN-γ or TNF-α through the replacement of extracellular loops with peptide fragments from OprF of P. aeruginosa. OmpA/OprF chimeras were developed, capable of binding IFN-γ or TNF-α. The specific peptide fragments that bind IFN-γ were identified. IFN-γ or TNF-α binding the OmpA/OprF chimera induced the pspA promoter, driving β-galactosidase production. The OmpA/OprF chimera had a detection limit of 300 pM for IFN-γ and 150 pM for TNF-α. This work will further the development of bacteria based therapeutics for the treatment of inflammatory diseases of the gut.
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, 14853, New York. jcm224@cornell.edu.
authors.forename: T Christopher
authors.initials: TC
authors.lastname: Aurand
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, 14853, New York.
authors.forename: John C
authors.initials: JC
authors.lastname: March
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism*; D062165:Receptors, Artificial / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C547605:OprF protein, Pseudomonas aeruginosa; D062165:Receptors, Artificial; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C078167:OMPA outer membrane proteins; D007371:Interferon-gamma
keywords: OmpA; OmpA OprF chimera; OprF; interferon-γ; pspA; tumor necrosis factor-α