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. 2022 Nov 28;11(23):3850.
doi: 10.3390/foods11233850.

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

Wanqiu Xia  1 , Jing Liu  1 , Jianping Wang  1
Affiliations

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

Wanqiu Xia et al. Foods. .

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.

Keywords: TetR; fluorescence polarization assay; milk; mutant; recognition mechanism; tetracyclines.

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Conflict of interest statement

All of the authors declare that they have no conflicts of interest. This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with humans. All the authors listed in the manuscript are entitled to the authorship and have approved the final version of the submitted manuscript.

Figures

Figure 1
Figure 1
(A) Synthetic routs and (B) UV scanning diagrams of FITC-MC and FA-MC.
Figure 2
Figure 2
(A) The 3D structure of the natural TetR, (B) its binding pocket, and (C) its docking complex with DMC. (D) The 3D structure of the mutant, (E) its binding pocket, and (F) its docking complex with DMC.
Figure 3
Figure 3
(A) Agarose gel electrophoresis results of (a) The TetR mutant (containing 6 × His-tag, about 170 bp) and (b) express vector pET28a-TetR mutant. (B) SDS-PAGE result of the TetR mutant (lane 1, bacterial whole protein; lane 2, purified inclusion body; lane 3, purified supernatant). (C) Western blotting result of the TetR mutant.
Figure 4
Figure 4
FP values when testing DMC and other drugs by using the two fluorescent tracers (DI = diazepam, ST = streptomycin, CI = ciprofloxacin, CH = chlorpromazine; n = 5).
Figure 5
Figure 5
Competitive curves of DMC when using the natural TetR and the mutant (n = 5).
Figure 6
Figure 6
Change trends of the binding energy, absolute affinity constant (KA), and IC50 for the 10 TCs when using the natural TetR and the mutant.
See this image and copyright information in PMC

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References

    1. Nelson M.L., Levy S.B. The history of the tetracyclines. Ann. N. Y. Acad. Sci. 2011;1241:17–32. doi: 10.1111/j.1749-6632.2011.06354.x. - DOI - PubMed
    1. Griffin M.O., Fricovsky E., Ceballos G., Villarreal F. Tetracyclines: A pleitropic family of compounds with promising therapeutic properties. Review of the literature. Am. J. Physiol. Cell Ph. 2010;299:C539–C548. doi: 10.1152/ajpcell.00047.2010. - DOI - PMC - PubMed
    1. Levy S.B., McMurry L. Detection of an inducible membrane protein associated with R-factor-mediated tetracycline resistance. Biochem. Biophys. Res. Commun. 1974;56:1060–1068. doi: 10.1016/S0006-291X(74)80296-2. - DOI - PubMed
    1. Hillen W., Klock G., Kaffenberger I., Wray L.V., Reznikoff W.S. Purification of the TET repressor and TET operator from the transposons Tn10 and characterization of their interaction. J. Biol. Chem. 1982;252:6605–6613. doi: 10.1016/S0021-9258(20)65186-9. - DOI - PubMed
    1. Hinrichs W., Kisker C., Düvel M., Müller A., Tovar K., Hillen W., Saenger W. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science. 1994;264:418–420. doi: 10.1126/science.8153629. - DOI - PubMed

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