Synthesis of photoaffinity labeled activity-based protein profiling probe and production of natural TetR protein for immunoassay of tetracyclines in milk

Production of a Natural Dihydropteroate Synthase and Development of a Signal-Amplified Pseudo-Immunoassay for the Determination of Sulfonamides in Pork

“…It is well-known that the receptor–ligand binding is based on noncovalent bonds, so the natural DHPS could be easily obtained by eluting the probe/DHPS complex with methanol/PBS (3:7, v/v), just like eluting the absorbed analytes from an immunoaffinity chromatography column . Compared to the conventional ABPP probe, the use of magnetic ABPP probe to produce a receptor was much simpler, more rapid, and cheaper.…”

“…However, all of the previously used DHPS proteins for the analysis of SAs are the recombinant products that are expressed according to the genes of previous studies, so they should have some differences from the natural DHPS proteins. It is well-known that the production of a natural receptor using a conventional procedure is tedious and expensive, and the obtained product has many uncertainties. , In comparison, the use of photoaffinity-labeled activity-based protein-profiling probe (PAL-ABPP) to produce a natural receptor is simple and cheap, so this type of probe has been used to study the receptors of some small-molecule substances. , In our recent report, a type of PAL-ABPP was synthesized; it was used to capture the natural TetR protein from a tetracycline-resistant bacteria, and the obtained receptor simultaneously recognized all of the tested tetracycline drugs with comparable sensitivities …”

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

“…The amplified gene was inserted into pET28a to construct the express vector that was transformed into E. coli Rosetta-gami(BL21) to express the mutant, and these procedures were according to our recent report [ 30 ]. The obtained TetR mutant was characterized by using SDS-PAGE and western blotting according to our recent report [ 36 ], and its recognition ability for the 10 TCs was evaluated by the following FPA method. Furthermore, its recognition mechanisms and affinities for the 10 TCs were also determined as described above.…”

“…In our recent study, a type of natural TetR protein capable of recognizing 10 TCs was obtained [ 36 ], but its identity was not identified and its recognition mechanisms for TCs are unknown. In the present study, its identity was characterized by using the LC-ESI-MS/MS technique, and its recognition mechanisms and affinities for the 10 TCs shown in Table 1 were thoroughly studied by using molecular docking and surface plasmon resonance, respectively.…”

Development of a Fluorescence Polarization Assay for Multi-Determination of 10 Aminoglycosides in Pork Muscle Sample Based on Ribosomal Protein S12 and Studying Its Recognition Mechanism

“…It is well known that the receptor is a natural macromolecule, and a specific receptor is the usual target site of one class of drugs, so in theory, it should recognize all the species of this class of drugs. During the past few years, some receptors have been used as recognition reagents to develop pseudo-immunoassays for the detection of sulfonamides [ 19 , 20 , 21 , 22 , 23 ], β-lactams [ 24 ], tetracyclines [ 25 , 26 , 27 ], and β 2 -agonists [ 28 ]. Results showed that these receptors exhibited broader detection spectra than the controlled antibodies.…”