Induction of TSH binding inhibitory immunoglobulins with the extracellular domain of human thyrotropin receptor produced using baculovirus expression system.
A cDNA encoding the extracellular domain of human TSHr (ETSHr) was expressed in large quantities using the baculovirus expression system. Maximum level of protein was produced at 60 hr post-infection and represented approximately 20% of the total cellular protein. The identity of the protein as ETSHr was confirmed by Western blot using antibodies to synthetic peptides derived from the TSHr. The protein has an apparent molecular weight of 50 kDa and is larger than the predicted size of 44 kDa, suggesting that the protein is glycosylated. Polyclonal antibodies raised in rabbits against gel purified ETSHr blocked the binding of 125I-TSH to native TSHr in solubilized porcine thyroid membranes in a radioreceptor assay. The availability of this antigenically active protein will facilitate further characterization of the protein and analysis of immune response against TSHr in experimental animals as well as in patients with autoimmune Graves' disease.
Seetharamaiah
G S
GS
Department of Microbiology, University of Texas Medical Branch, Galveston 77555.
Desai
R K
RK
Dallas
J S
JS
Tahara
K
K
Kohn
L D
LD
Prabhakar
B S
BS
D000818:Animals; D001323:Autoantibodies / Q000096:biosynthesis*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D018828:Immunoglobulins, Thyroid-Stimulating; D008969:Molecular Sequence Data; D009036:Moths; D011817:Rabbits; D011989:Receptors, Thyrotropin / Q000096:biosynthesis* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013961:Thyroid Gland / Q000276:immunology*
D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins