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קהילה:
אסיף מאגר המחקר החקלאי
פותח על ידי קלירמאש פתרונות בע"מ -
Large-scale preparation and characterization of recombinant ovine placental lactogen
Year:
1997
Source of publication :
Journal of Endocrinology
Authors :
שמאי, אבי
;
.
Volume :
152
Co-Authors:
Sakal, E., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Bignon, C., U. d'Endocrinol. Moleculaire, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Grosclaude, J., U. de Virologie et Immunol. Molec., Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Kantor, A., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Shapira, R., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Leibovitch, H., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Helman, D., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Nespoulous, C., U. Biochim. et Struct. des Proteines, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Shamay, A., Animal Science Division, Agricultural Research Organization, Rehovot 76100, Israel
Rowlinson, S.W., Dept. of Physiology and Pharmacology, Centre of Molecular and Cell Biology, University of Queensland, St Lucia, QLD 4072, Australia
Djiane, J., U. d'Endocrinol. Moleculaire, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Gertler, A., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Facilitators :
From page:
317
To page:
327
(
Total pages:
11
)
Abstract:
To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-β-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate β-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.
Note:
Related Files :
Animals
animal tissue
Cell Proliferation
Female
gene expression
placenta lactogen
sheep
עוד תגיות
תוכן קשור
More details
DOI :
Article number:
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
22627
Last updated date:
02/03/2022 17:27
Creation date:
16/04/2018 23:53
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Scientific Publication
Large-scale preparation and characterization of recombinant ovine placental lactogen
152
Sakal, E., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Bignon, C., U. d'Endocrinol. Moleculaire, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Grosclaude, J., U. de Virologie et Immunol. Molec., Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Kantor, A., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Shapira, R., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Leibovitch, H., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Helman, D., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Nespoulous, C., U. Biochim. et Struct. des Proteines, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Shamay, A., Animal Science Division, Agricultural Research Organization, Rehovot 76100, Israel
Rowlinson, S.W., Dept. of Physiology and Pharmacology, Centre of Molecular and Cell Biology, University of Queensland, St Lucia, QLD 4072, Australia
Djiane, J., U. d'Endocrinol. Moleculaire, Inst. Natl. de la Rech. Agronomique, 78352 Jouy-en-Josas Cedex, France
Gertler, A., Inst. Biochem., Food Sci. and Nutr., Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
Large-scale preparation and characterization of recombinant ovine placental lactogen
To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-β-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate β-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.
Scientific Publication
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