Cell-free expression, purification, and characterization of the functional β2-adrenergic receptor for multianalyte detection of β-agonists

Abstract

Large-scale expression of β-adrenergic receptor (β-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 manycritical 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 β-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β-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 β-AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 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 β-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.

Expression of Codon Optimized ��2-Adrenergic Receptor in Sf9 Insect Cells for Multianalyte Detection of ��-Agonist Residues in Pork

“…The binding affinity of β-agonist to the purified β 2 -AR protein was verified first by a direct ELRA as described in our previous literature [27]. Then a competitive ELRA format was adopted for further analyzing β was used to stop the reaction and the absorbance was measured at 450 nm.…”

“…However, the method showed the potential of the receptor to bind a panel of structurally different β-agonists and new compounds with agonistic activity undetectable by the previous methods. Moreover, the established competitive ELRA exhibited better performance than the current ELRA methods [25][26][27][28], providing a great application prospect for multianalyte screening of abused and illegal β-agonist drugs in pork and other related animal products.…”

“…But owing to the large costs and possible harmful impact of radioactive isotopes on the human body, it is imperative to develop nonradioactive detection methods at present. Our team successively established direct, competitive enzyme-linked receptor assay (ELRA) methods based on β 2 -AR protein synthetized in HEK293 cells [26] and a cell-free system [27] for multianalyte detection of β-agonists. In addition, Cheng et al [28] also developed an ELRA method using wild-type Syrian hamster β 2 -AR expressed in Sf9 cells to determine β-agonists in animal feed.…”