In this study, the identity of our recently produced natural TetR protein was identified by using the LC-ESI-MS/MS technique, and its recognition mechanisms, including the binding pocket, contact amino acids, intermolecular forces, binding sites, binding energies, and affinities for 10 tetracycline drugs were studied. Then, it was evolved by site-mutagenesis of an amino acid to produce a mutant, and a fluorescence polarization assay was developed to detect the 10 drugs in milk. The sensitivities for the 10 drugs were improved with IC50 values decreasing from 30.8–80.1 ng/mL to 15.5–55.2 ng/mL, and the limits of detection were in the range of 0.4–1.5 ng/mL. Furthermore, it was found that the binding affinity for a drug was the critical factor determining its sensitivity, and the binding energy showed little influence. This is the first study reporting the recognition mechanisms of a natural TetR protein for tetracyclines and the development of a fluorescence polarization assay for the detection of tetracyclines residues in food samples.
In this study, the genes of Escherichia coli ribosomal protein S12 and renilla luciferase were linked and expressed to produce a fusion protein, and its intermolecular interactions and affinities with sevenaminoglycosides were studied. Then, the fusion protein was used as the core agent to develop a bioluminescent method on a conventional microplate for determination of the residues of thesevenaminoglycosides in pork. This method contained only one sample-loading step, and thus the assay was finished within 30 min. The limits of detection for the sevendrugs were in the range of 0.51–1.1 ng/mL, and the sensitivity for a specific drug was mainly determined by the receptordrug affinity but not related with the binding energy. After general comparison, the present method showed generally better performances than the previously reported enzyme-linked immunosorbent assays for aminoglycosides. This is the first study reporting the recognition mechanisms of Escherichia coli ribosomal protein S12 for aminoglycosides and developing a bioluminescent method for detection of aminoglycoside residues in pork samples.
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