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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. [ABSTRACT FROM AUTHOR]

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillussphaericus) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillussphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples. [ABSTRACT FROM AUTHOR]

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.

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillussphaericus) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillussphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples.