Characterization of soluble, disulfide bond-stabilized, prokaryotically expressed human thyrotropin receptor ectodomain.
To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein; it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) 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 transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR 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 (strain alpha F'), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.
Bobovnikova
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Y
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Graves
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PN
Vlase
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H
Davies
T F
TF
D000906:Antibodies / Q000032:analysis; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000276:immunology; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013972:Thyrotropin / Q000378:metabolism
D000906:Antibodies; D004220:Disulfides; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin