Expression of a biotinylated human thyrotropin receptor in HeLa cells using recombinant vaccinia virus and its application for the detection of Graves' autoantibodies.
We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).
Minich
W B
WB
Department of Internal Medicine I, University of Ulm, Germany.
Weymayer
J D
JD
Loos
U
U
D001323:Autoantibodies / Q000032:analysis*; D001426:Bacterial Proteins / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D005822:Genetic Vectors; D006111:Graves Disease / Q000276:immunology*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D015290:Second Messenger Systems / Q000502:physiology; D012685:Sepharose / Q000031:analogs & derivatives / Q000494:pharmacology; D013972:Thyrotropin / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics*
D001323:Autoantibodies; D001426:Bacterial Proteins; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; C049538:streptavidin-agarose; D001710:Biotin; D013972:Thyrotropin; D012685:Sepharose; D000242:Cyclic AMP