Cell-free expression, purification, and characterization of the functional β2-adrenergic receptor for multianalyte detection of β-agonists
- Authors: Wang J.1,2, Liu Y.1,3, Zhang J.3, Han Z.1, Wang W.1, Liu Y.1, Wei D.1, Huang W.3
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Affiliations:
- Food Safety Research Center
- Institute of Quality Standards and Testing Technology for Agro-Products of CAAS, Key Laboratory of Agro-Product Quality and Safety
- Hebei North University
- Issue: Vol 82, No 11 (2017)
- Pages: 1346-1353
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151514
- DOI: https://doi.org/10.1134/S0006297917110128
- ID: 151514
Cite item
Abstract
Large-scale expression of β2-adrenergic receptor (β2-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have many critical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β2-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β2-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β2-AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC50 values were 45.99, 60.38, and 78.02 μg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.
About the authors
Jian Wang
Food Safety Research Center; Institute of Quality Standards and Testing Technology for Agro-Products of CAAS, Key Laboratory of Agro-Product Quality and Safety
Author for correspondence.
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000; Beijing, 100081
Yuan Liu
Food Safety Research Center; Hebei North University
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000; Zhangjiakou, 075000
Junhua Zhang
Hebei North University
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000
Zhengzheng Han
Food Safety Research Center
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000
Wei Wang
Food Safety Research Center
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000
Yang Liu
Food Safety Research Center
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000
Dong Wei
Food Safety Research Center
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000
Wei Huang
Hebei North University
Email: xuanyuanjian0228@126.com
China, Zhangjiakou, 075000