A simple quick and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies

摘要

Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by several different detergents for comparison, and was finally purified to a single band at a nearly perfect ratio of 1:0.9 ± 0.05 (ligand:receptor molecule) in ligand binding using a Ni-column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNA-digestion, DM detergent-extraction, centrifugation and FPLC-purification. The purity and quantity of the purified TP, using the high-yield approach, was suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution 1D and 2D ¹H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.