CHARACTERIZATION OF SOLUBLE, DISULFIDE BOND-STABILIZED, PROKARYOTICALLY EXPRESSED HUMAN THYROTROPIN RECEPTOR ECTODOMAIN

To study the interaction of TSH receptor (TSHR) autoantibodies with re ceptor protein, it is necessary first to express the receptor in the p roper conformation including the formation of correct disulfide bridge s. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limi ts the solubility and immunoreactivity of recombinant human TSHR (hTSH R) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21-415) was inserted into the vector pGEX-2TK by directional cloning and used to t ransform the thioredoxin reductase mutant strain of E. coli (Ad494), w hich allowed formation of disulfide bonds in the cytoplasm. After indu ction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTS HR amino acids 21-35. This showed that over 50% of the expressed hTSHR -ecd was soluble in contrast to expression in a wild-type E. coli (str ain (alpha F'), where the majority of the recombinant receptor was ins oluble. The soluble recombinant receptor was affinity purified and cha racterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-e cd migrated as refolded, disulfide bond-stabilized, multimeric species , whose formation was independent of fusion partner protein. This prod uct was found to be biologically active as evidenced by the inhibition of the binding of I-125-TSH to the full-length hTSHR expressed in tra nsfected CHO cells and was used to develop a competitive capture enzym e-linked immunosorbent assay for mapping of hTSHR antibody epitopes. H ence, hTSHR-ecd produced in bacteria with a thioredoxin reductase muta tion was found to be highly soluble and biologically relevant.