OVEREXPRESSION OF THE EXTRACELLULAR DOMAIN OF THE THYROTROPIN RECEPTOR IN BACTERIA - PRODUCTION OF THYROTROPIN-BINDING INHIBITING IMMUNOGLOBULINS

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requir es 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-cR1 vector. The major protein in an electrophoretically separ ated, crude bacterial lysate had a molecular mass of 89 kDa, in agreem ent with the size predicted for the MBP-ECD fusion product. Its identi ty was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-M BP. Following purification on an amylose column, 15 mg pure MBP-ECD pe r litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces sligh t denaturation, MBP-ECD was cleaved with factor Xa. The identity of ea ch protein was confirmed by Western blotting. To investigate the possi bility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluo rescent staining of Chinese hamster ovary cells that expressed the TSH R, 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 d etected in Western blots of human thyroid membranes. Subsequently, imm unoglobulins from mice immunized with MBP-ECD were shown to contain TS H-binding inhibiting activity and to inhibit TSH-mediated cyclic AMP p roduction; these mice had a lower serum thyroxine level when compared with mice immunized with the MBP-beta galactosidase fusion protein MBP -GAL. The study shows the feasibility of using recombinant TSHR expres sed in E. coli (i) to produce antibodies which recognize the native re ceptor and thus could be applied to studies of TSHR expression (e.g. i n thyroid tumours), (ii) to establish animal models of autoimmune hypo thyroidism and (iii) as the starting material in denaturation and refo lding experiments which may help in defining structure-function relati onships.