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 expression of the receptor was confirmed by immunocytochemistry with polyclonal antibodies raised against the extracellular domain of the receptor. The expressed receptor was functional; both thrombin and the thrombin receptor agonist peptide produced increases in intracellular calcium in transfected cells. The concentration of thrombin causing the half-maximal increase (EC50) in intracellular calcium was 3.9 nM, whereas the EC50 for the agonist peptide was 2.7 microM. However, the observed maximum increase in intracellular calcium concentration with the agonist peptide (547 nM) was twofold greater than that observed with thrombin (258 nM). The recombinant receptor was purified by immunoaffinity chromatography using a monoclonal antibody raised against the receptor extracellular domain. The purified preparation contained two species with apparent molecular masses of 48 and 90 kDa, both of which were recognized by mono- and polyclonal antibodies against the thrombin receptor. The yield of the purified receptor was 0.78 mg/liter of insect cells suspension culture (10(6) cells/ml). The purified thrombin receptor will be useful in future structural and functional studies.
Chinni
C
C
Department of Haematology, MRC Centre, University of Cambridge, United Kingdom. carla.chinni@med.monash.edu.au
Bottomley
S P
SP
Duffy
E J
EJ
Hemmings
B A
BA
Stone
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018179:Receptors, Thrombin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D002118:Calcium