The expression of soluble, full-length, recombinant human TSH receptor in a prokaryotic system.
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.
Busuttil
B E
BE
Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin and Repatriation Medical Centre, The University of Melbourne, Austin Campus, Heidelberg, Victoria, 3084, Australia.
Turney
K L
KL
Frauman
A G
AG
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006111:Graves Disease / Q000276:immunology; D006639:Histidine / Q000378:metabolism; D006801:Humans; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D001323:Autoantibodies; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D006639:Histidine