A simple, quick, and high-yield preparation of the human thromboxane A2 receptor in full size for structural studies.
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 different detergents for comparison and was finally purified to a nearly single band with a 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, dodecyl-maltoside detergent extraction, centrifugation, and FPLC purification. The purity and quantity of the purified TP, using the high-yield approach, were suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution one-dimensional and two-dimensional (1)H 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.
Ruan
Ke-He
KH
Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, Houston, Texas 77204-5037, USA. khruan@uh.edu
Cervantes
Vanessa
V
Wu
Jiaxin
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera; D013997:Time Factors
D018076:DNA, Complementary; D008024:Ligands; D044244:Receptors, Thromboxane A2, Prostaglandin H2