Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease.
Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.
Huang
G C
GC
Department of Medicine, King's College School of Medicine and Dentistry, Denmark Hill, London.
Collison
K S
KS
McGregor
A M
AM
Banga
J P
JP
D001323:Autoantibodies / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space; D005982:Glutathione Transferase / Q000235:genetics; D006111:Graves Disease / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*
D001323:Autoantibodies; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin; D004247:DNA; D005982:Glutathione Transferase