Overexpression of the extracellular domain of the thyrotrophin receptor in bacteria; production of thyrotrophin-binding inhibiting immunoglobulins.
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-cR1 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Costagliola
S
S
IRIBHN, Université Libre de Bruxelles, Hôpital Erasme, Belgium.
Alcalde
L
L
Ruf
J
J
Vassart
G
G
Ludgate
M
M
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis*; D008297:Male; D058619:Maltose-Binding Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011989:Receptors, Thyrotropin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013961:Thyroid Gland / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007136:Immunoglobulins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli