Expression of the functional extracellular domain of human thyrotropin receptor using a vaccinia virus system: its purification and analysis of autoantibody binding.
We produced substantial amount of the extracellular domain of the human TSH receptor (TSHRE) that has a tag of six histidines at C-terminus as a soluble form in the human cell line HeLa using a vaccinia virus system. By sequential nickel-chelating and lentil lectin column chromatography, TSHRE was purified to about 70% purity, with the recovery of around 0.1-0.2 mg TSHRE/L culture (5 x 10(8) cells/liter culture). The purified TSHRE interacted with TSH as well as Graves' autoantibodies to TSHR. However, the affinity of TSHRE for TSH was much lower than that of intact TSHR. The IC50 value for inhibition of TSH-dependent cAMP synthesis by TSHRE was about 10(-8) mol/L. Most importantly, the purified TSHRE inhibited the binding of the IgG of Graves' patients to thyroid membrane. About 1 microg/mL (2 x 10(-8) mol/L) TSHRE neutralized most of the autoantibody activity of patients' sera tested in the TSH binding inhibitory immunoglobulin (TBII) assay. Moreover, this protein neutralized thyroid stimulatory antibody-induced cAMP synthesis with an IC50 of 1 x 10(-9) mol/L and completely at 0.5-1 microg/mL (1-2 x 10(-8) mol/L). In the simple enzyme-linked immunosorbent assay, the TSHRE immobilized on the wells coated with nickel showed significantly higher binding with the IgGs from Graves' patients than in those from normal individuals. This autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and the development of therapy of Graves' disease.
Lee
M H
MH
Department of Life Science and Biotechnology Research Center, Pohang University of Science and Technology, Korea.
Park
J Y
JY
Cho
B Y
BY
Chae
C B
CB
D001323:Autoantibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006111:Graves Disease / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D011989:Receptors, Thyrotropin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D013972:Thyrotropin / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics
D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin