A RECOMBINANT EXTRACELLULAR DOMAIN OF THE THYROTROPIN (TSH) RECEPTOR BINDS TSH IN THE ABSENCE OF MEMBRANES

We produced large quantities of the extracellular domain of the human TSH receptor (ETSHR) using the baculovirus expression system. Insect c ells containing the ETSHR protein were sequentially extracted using ly sis, nuclease, and high salt buffers to enrich for recombinant protein . The ETSHR protein was purified to homogeneity on a C4 reverse phase semipreparative column using HPLC. The recombinant protein was identif ied as ETSHR by immunoreactivity with antibodies prepared against TSHR -derived synthetic peptides. The identity of the ETSHR was further con firmed by amino acid compositional analyses, which agreed with the ami no acid composition predicted from reported cDNA sequence analyses. Pr otein sequence analyses confirmed that the first 26 amino acids of the N-terminal region and the C-terminal amino acid were identical to the predicted amino acid sequence. The purified ETSHR was refolded in the presence of 1.5 M guanidine-HCl and 1 mM each of cystine and cysteine . [I-125] TSH bound to the refolded ETSHR in vitro in a dose-dependent manner and was specifically blocked by unlabeled TSH, but not by LH o r FSH. It was notable that a membrane requirement was not essential TS H to bind to ETSHR.