Site-specific immobilization of Cysteinyl leukotriene receptor 1 through enzymatic DNA-protein conjugation strategy for lead screening.

Autor: Wen X; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China., Chen M; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China., Li Z; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China., Liu W; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China., Xu K; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China., Wang J; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China. Electronic address: waangjing@nwu.edu.cn., Zhao X; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China.
Jazyk: angličtina
Zdroj: Journal of chromatography. A [J Chromatogr A] 2024 Jul 19; Vol. 1727, pp. 464948. Date of Electronic Publication: 2024 Apr 27.
DOI: 10.1016/j.chroma.2024.464948
Abstrakt: Immobilization of functional protein, especially G protein-coupled receptors (GPCRs), is particularly significant in various fields such as the development of assays for diagnosis, lead compound screening, as well as drug-protein interaction analysis. However, there are still some challenges with the immobilized proteins such as undefined loads, orientations, and the loss of activity. Herein, we introduced a DNA conjugation strategy into the immobilization of Cysteinyl leukotriene receptor 1(CysLTR1) which enables exquisite molecular control and higher activity of the receptor. We used the bacterial relaxases VirD2 as an immobilized tag fused at the C terminus of CysLTR1. Tyrosine residue(Y29) at the core binding site of the VirD2 tag can react with the single-strand piece of DNA(T-DNA) in the form of a covalent bond. Inspired by this strategy, we developed a new immobilization method by mixing the T-DNA-modified silica gel with the cell lysate containing the expressed VirD2-tagged CysLTR1 for 1 hour. We found that the successful formation of DNA-protein conjugate enables the immobilization of CysLTR1 fast, site-specific, and with minimal loss of activity. The feasibility of the immobilized CysLTR1 was evaluated in drug-protein binding interaction by frontal analysis and adsorption energy distribution analysis. The binding of pranlukast, zafirlukast, and MK571 to the immobilized CysLTR1 was realized, and the association constants presented good agreement between the two methods. Rosmarinic acid was retained in the immobilized CysLTR1 column, and the in-vitro test revealed that the compound binds to the receptor in one type of binding site mode. Despite these results, we concluded that the DNA-protein conjugate strategy will probably open up the possibilities for capturing other functional proteins in covalent and site-specific modes from the complex matrices and the immobilized receptor preserves the potential in fishing out lead compounds from natural products.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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