Folding-Dependent Binding of Thyrotropin (TSH) and TSH Receptor Autoantibodies to the Murine TSH Receptor Ectodomain

The mouse TSH receptor ectodomain (mTSHR-ecd) was amplified from murine thyroid complementary DNA and ligated into the pAcGP67B insect cell vector, and the nucleotide sequence was confirmed. Employing a baculovirus-insect cell system, the mTSHR-ecd (amino acids 22–415) was expressed as a fusion protein with the gp67 insect cell signal sequence at the NH2-terminus and a C-terminal six-histidine tag. Protein expression was assessed by Western blot using a murine monoclonal antibody (recognizing amino acids 22–35) and a rabbit antipeptide antibody (recognizing amino acids 397–415). These antibodies detected two principal species of mTSHR-ecd, one glycosylated (66 kDa) and one nonglycosylated (52 kDa), in cell lysates of infected insect cells. More than 10% of these species were present in a water-soluble (cytosolic) fraction. This fraction was then used to purify, under native conditions, 100-μg amounts of mTSHR-ecd using nickel-nitrilo-triacetic (Ni-NTA) resin chromatography. The purified cytosolic mTSHR-ec...