Identification and Evolution of a Natural Tetr Protein Based on Molecular Docking and Development of a Fluorescence Polari-Zation Assay for Multi-Detection of 10 Tetracyclines in Milk

Abstract

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.

Molecular simulation for food protein–ligand interactions: A comprehensive review on principles, current applications, and emerging trends

“…Biotoxins like aflatoxin B1 (Rahi et al, 2023), residues of pesticides like procymidone (Liu et al, 2023), and veterinary drugs like tetracyclines (Xia et al, 2023) have been explored by molecular docking, according to recent reports. In the research of Zhou, Lan et al ( 2023), molecular docking between Bacillus cereus-specific aptamers and proteins was performed.…”

Fluorescence Polarization Assay Based on a New Recognition Motif QepA for the One-Step Detection of Fluoroquinolones in Eggs

“…To date, several papers have utilized receptors as recognition elements in the detection of specific analytes. For instance, TetR has been employed to detect tetracyclines, − dihydropteroate synthase (DHPS) has been used to detect sulfonamides, ,− and penicillin-binding proteins (PBPs) have been used to detect β-lactams. , Therefore, enzymes, targets, and proteins with specific recognition and binding abilities to compounds show potential to be used as alternatives to traditional antibodies in the rapid detection of contaminants. According to the knowledge of pharmacology and microorganism resistance, gyrase, topoisomerase IV (TopIV), and QepA have the ability to bind with FQs.…”