Generation of epitope-specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site-directed mutagenesis of ovine GHBP.
Site-directed antibodies to the growth hormone receptor could be potentially useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope-switching between rat and ovine growth hormone receptors to produce rat epitopes in the correct structural context. Using site-directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34, is equivalent to epitope 1 in ovine growth hormone binding protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild-type ovine growth hormone binding protein and the two mutant proteins were bacterially expressed, refolded and, following purification by metal-chelate affinity chromatography, used to raise antisera in sheep. We showed using RIA, in which wild-type ovine growth hormone binding protein acted as a competitor for the binding of rat growth hormone binding protein, that only the site A mutant protein elicited a specific anti-rat growth hormone binding protein response. This was confirmed in subsequent RIA studies using the antiserum to the site A mutant protein in which only peptides corresponding to the site A sequences in mutant ovine growth hormone binding protein and rat growth hormone binding protein, but not that in wild-type ovine growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild-type ovine growth hormone binding protein to remove antibodies which cross-reacted with the ovine protein. The work lays the foundations for further studies in which the biological effects of these antibody fractions will be investigated and demonstrates an approach with general applicability in the production of antibodies directed towards specific epitopes on protein molecules.
Shand
J H
JH
Hannah Research Institute, Ayr, UK. shandj@hri.sari.ac.uk
Allan
G J
GJ
Beattie
J
J
Flint
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D013006:Growth Hormone / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011863:Radioimmunoassay; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012756:Sheep; D013045:Species Specificity
D002352:Carrier Proteins; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; C051815:somatotropin-binding protein