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

详细

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.