EXPRESSION AND PURIFICATION OF THE HUMAN THROMBIN RECEPTOR

The human thrombin receptor has been overexpressed in Sf9 (Spodoptera frugiperda) insect cells using a baculovirus vector. Cell surface expr ession of the receptor was confirmed by immunocytochemistry with polyc lonal antibodies raised against the extracellular domain of the recept or. The expressed receptor was functional; both thrombin and the throm bin receptor agonist peptide produced increases in intracellular calci um in transfected cells. The concentration of thrombin causing the hal f-maximal increase (EC50) in intracellular calcium was 3.9 nM, whereas the EC50 for the agonist peptide was 2.7 mu M. However, the observed maximum increase in intracellular calcium concentration with the agoni st peptide (547 nM) was twofold greater than that observed with thromb in (258 nM). The recombinant receptor was purified by immunoaffinity c hromatography using a monoclonal antibody raised against the receptor extracellular domain. The purified preparation contained two species w ith apparent molecular masses of 48 and 90 kDa, both of which were rec ognized by mono-and polyclonal antibodies against the thrombin recepto r. The yield of the purified receptor was 0.78 mg/liter of insect cell s suspension culture (10(6) cells/ml). The purified thrombin receptor will be useful in future structural and functional studies. (C) 1998 A cademic Press.