Directional evolution of TetR protein and development of a fluoroimmunoassay for screening of tetracyclines in egg

Abstract

In this study, the contact amino acid Pro105 in the binding pocket of TetR protein was directly substituted with Phe and Tyr to produce the mutant1 and the mutant2 respectively. The two mutants simultaneously recognized 9 tetracycline drugs, and they showed highly increased affinities to the 9 drugs with 1.5–13.3 folds of increased sensitivity in comparison with the parental TetR. The molecular docking revealed that the mutant2 with improved stability showed higher sensitivity to the 9 drugs than the mutant1 with highly increased binding energy. Then the mutant2 was combined with a fluorescent-labeled tracer to develop a one-step competitive fluoroimmunoassay for simultaneous screening of the 9 drugs in egg. The tracer and the sample extract were added into the mutant2-coated microplate wells for competition, and the fluorescence intensity was excited directly for analyte quantification. Results showed that the half of inhibition concentrations for the 9 drugs were in the range of 3.1–17.2 ng/mL, and the limits of detection were in the range of 0.3–5.8 ng/mL. Therefore, this method could be used as a routine screening tool to monitor the residues of the 9 tetracyclines in egg.