Expression of a functional tagged human thyrotropin receptor in HeLa cells using recombinant vaccinia virus.
We report a new effective system for expression of FLAG and six histidine tagged TSH receptor in mammalian cells using recombinant vaccinia virus. HeLa cells infected with recombinant virus produced large amounts of human TSH receptor of approximately 150,000 functional molecules per cell. This value is one to two orders of magnitude higher than those in thyroid cells and is comparable with receptor number of the best stably transfected mammalian cell clones previously described. Vaccinia virus produced TSH receptor was able to bind TSH (Kd of 2.1 +/- 0.1 x 10(-10) M) and Graves'disease autoantibodies. TSH caused an increase of the intracellular cAMP level in infected HeLa cells in a concentration-dependent manner, demonstrating the coupling of expressed recombinant TSH receptor to the cAMP second messenger system of the cells. 6His-tagged recombinant TSH receptor was immobilized on Ni2+ nitrilotriacetic acid-agarose. Bound receptor was fully functional, interacting with both TSH and Graves' disease autoantibodies in patient sera. The solid phase bound TSH receptor technique provides a new and simple method for the diagnosis of autoimmune diseases.
Minich
W B
WB
Department of Internal Medicine I, University of Ulm, Germany.
Behr
M
M
Loos
U
U
D001323:Autoantibodies / Q000276:immunology; D001327:Autoimmune Diseases / Q000175:diagnosis; D000242:Cyclic AMP / Q000378:metabolism; D006111:Graves Disease / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D016015:Logistic Models; D011989:Receptors, Thyrotropin / Q000032:analysis*; D011995:Recombination, Genetic; D015290:Second Messenger Systems / Q000502:physiology; D014615:Vaccinia / Q000276:immunology / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics*
D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP