Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. Application to the human thyrotropin receptor.
The clinical applicability of a newly described polymerase chain reaction directed protein expression system was assessed for the in vitro synthesis and partial epitope mapping of large radiolabeled human thyrotropin receptor (hTSH-R) protein segments. PCR amplification of targeted regions within the hTSH-R cDNA followed by in vitro transcription and translation permitted rapid synthesis of protein segments ranging in size from 18 to 62 kDa. Initial epitope mapping was directed at a 52 amino acid segment unique to the hTSH-R compared to otherwise homologous glycoprotein hormone receptors. Sera from Graves' disease patients known to have autoantibodies against the hTSH-R were used to immunoprecipitate two protein fragments differing only by the presence of the unique region in the larger fragment (E5) but not in the smaller fragment (E4). Dense precipitation bands were obtained using Graves' sera to immunoprecipitate E5 whereas little or no specific immunoprecipitation of E4 occurred. Normal sera gave only weak immunoprecipitation bands of E5. The technique provides significant advantages over conventional cloning methods and should have general applicability in the study of other protein targets of autoimmune disease.
Burch
H B
HB
Department of Medicine, Walter Reed Army Medical Center, Washington, DC 20307-5001.
Nagy
E V
EV
Kain
K C
KC
Lanar
D E
DE
Carr
F E
FE
Wartofsky
L
L
Burman
K D
KD
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000096:biosynthesis / Q000737:chemistry*; D001483:Base Sequence; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000276:immunology*; D011994:Recombinant Proteins
D001324:Autoantigens; D000939:Epitopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins