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For the first time soluble, full-length, recombinant, human thyroid-stimulating hormone (TSH) receptor (TSHR) has been expressed in a prokaryotic system. The full-length TSHR cDNA, obtained from normal human thyroid, was cloned into a pQE-9 vector, sequenced, and confirmed to be identical to the published sequence, to be full length, and to be in frame. Expression of the receptor was as a fusion protein with a hexahistidine tail at the amino terminal, in an Escherichia coli expression system. Approximately 2.5 mg of protein per liter of bacterial culture was recovered from the cell homogenate, after a single passage through a nickel-nitrilotriacetic acid resin column. An estimated 60% increase in purity of a band of expected size, 87 kDa, was observed upon gel electrophoresis and staining with Coomassie blue, after the single purification step. Immunoreactivity of the 87-kDa protein with Graves' sera was confirmed by Western blotting. |
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