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Reicher, S., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Niv-Spector, L., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Gertler, A., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Gootwine, E., Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
The wild-type (WT) GH2-N ovine growth hormone (oGH) and duplicated GH2-Z genes differ in their open reading frame by two nonsynonymous substitutions, predicting a two-amino-acid difference in their product (G9R/G63S). Three recombinant oGH muteins: G9R, G63S and G9R/G63S, were prepared by site-directed mutagenesis of the WT oGH gene, expressed in E. coli, refolded and purified as monomers with over 98% homogeneity. Gel-filtration experiments with WT oGH and the three muteins indicated formation of 1:2 complexes with oGH receptor extracellular domain (oGHR-ECD). Interactions of oGHR-ECD with the WT and the muteins were studied by surface plasmon resonance. Kinetics constants calculated using a two-site model predicted that G9R/G63S has the highest affinity to oGHR-ECD, WT oGH the lowest, and G9R and G63S have intermediate affinities. These relative affinities were further investigated by radioreceptor assay with EC50 values were the lowest for G9R/G63S, highest for WT oGH, and intermediate for G9R and G63S. Bioactivity of the WT oGH and oGH muteins was determined by proliferation assay with FDC-P1-3B9 cells stably transfected with rabbit GHR. Relative proliferation rates of cells in cultures treated with the WT, G63S, G9R or G9R/G63S variants were 100%, 183%, 259% and 498%, respectively. In COS-7 transfected with oGHR, LHRE-TK-luciferase and β-galactosidase plasmids G9R/G63S showed 18% higher activity than WT oGH (P < 0.001). Thus the product of the oGH duplicated copy has higher affinity for GHR and higher somatogenic activity. As the GH2-Z gene copy is expressed in the placenta, allelic differences at the oGH locus may influence feto-placental development. © 2007 Elsevier Inc. All rights reserved.
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Pituitary and placental ovine growth hormone variants differ in their receptor-binding ability and in their biological properties
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Reicher, S., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Niv-Spector, L., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Gertler, A., Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot, 76100, Israel
Gootwine, E., Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
Pituitary and placental ovine growth hormone variants differ in their receptor-binding ability and in their biological properties
The wild-type (WT) GH2-N ovine growth hormone (oGH) and duplicated GH2-Z genes differ in their open reading frame by two nonsynonymous substitutions, predicting a two-amino-acid difference in their product (G9R/G63S). Three recombinant oGH muteins: G9R, G63S and G9R/G63S, were prepared by site-directed mutagenesis of the WT oGH gene, expressed in E. coli, refolded and purified as monomers with over 98% homogeneity. Gel-filtration experiments with WT oGH and the three muteins indicated formation of 1:2 complexes with oGH receptor extracellular domain (oGHR-ECD). Interactions of oGHR-ECD with the WT and the muteins were studied by surface plasmon resonance. Kinetics constants calculated using a two-site model predicted that G9R/G63S has the highest affinity to oGHR-ECD, WT oGH the lowest, and G9R and G63S have intermediate affinities. These relative affinities were further investigated by radioreceptor assay with EC50 values were the lowest for G9R/G63S, highest for WT oGH, and intermediate for G9R and G63S. Bioactivity of the WT oGH and oGH muteins was determined by proliferation assay with FDC-P1-3B9 cells stably transfected with rabbit GHR. Relative proliferation rates of cells in cultures treated with the WT, G63S, G9R or G9R/G63S variants were 100%, 183%, 259% and 498%, respectively. In COS-7 transfected with oGHR, LHRE-TK-luciferase and β-galactosidase plasmids G9R/G63S showed 18% higher activity than WT oGH (P < 0.001). Thus the product of the oGH duplicated copy has higher affinity for GHR and higher somatogenic activity. As the GH2-Z gene copy is expressed in the placenta, allelic differences at the oGH locus may influence feto-placental development. © 2007 Elsevier Inc. All rights reserved.
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