The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21–414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cRl vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting.
To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes. Subsequently, immunoglobulins from mice immunized with MBP-ECD were shown to contain TSH-binding inhibiting activity and to inhibit TSH-mediated cyclic AMP production; these mice had a lower serum thyroxine level when compared with mice immunized with the MBP—β galactosidase fusion protein MBP-GAL.
The study shows the feasibility of using recombinant TSHR expressed in E. coli (i) to produce antibodies which recognize the native receptor and thus could be applied to studies of TSHR expression (e.g. in thyroid tumours), (ii) to establish animal models of autoimmune hypothyroidism and (iii) as the starting material in denaturation and refolding experiments which may help in defining structure—function relationships.
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