Protein–DNA interaction-based detection of small molecules by employing Renilla luciferase fusion protein: Quantitative and generic measurement of tetracyclines with a Renilla luciferase-tagged Tet repressor protein

Abstract

Many transcriptional regulator proteins serve as molecular sensors, the function of which is based on an allosteric mechanism where the binding of small molecules or ions to the regulator protein controls its association or dissociation with the corresponding regulatory DNA region. This characteristic of transcriptional regulator proteins has been harnessed by humans to develop biosensors for detection of various chemicals and ions [1]. A well-studied genetic regulatory system is the Tn10-encoded tetracycline-inducible Tet repressor–operator (TetR–tetO)4 pair, where binding of tetracycline abolishes the TetR–tetO interaction by nine orders of magnitude [2,3]. The TetR–tetO system has been used successfully in inducible regulation of gene expression in many organisms [4], and it has also been used as an analytical tool to detect tetracyclines by reporter gene expression [5,6]. Recently, Weber and coworkers [7] described a TetR– tetO interaction-based in vitro assay where anti-His tag antibody was used to detect the binding of His–TetR to immobilized tetO. The in vitro assay simpliWes the measure-