Hostname: page-component-586b7cd67f-rcrh6 Total loading time: 0 Render date: 2024-11-28T03:59:13.613Z Has data issue: false hasContentIssue false
  • English
  • Français

The porcine glucocorticoid receptor: sequencing a 2.1 kb cDNA fragment and raising specific polyclonal antibodies for western blotting, immunoprecipitation and immunohistochemistry

Published online by Cambridge University Press:  18 August 2016

M. Gutscher ,
S. Eder ,
M. Müller  and
R. Claus
M. Gutscher
Affiliation:
Fachgebiet Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung (470a), Universität Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany
S. Eder
Affiliation:
Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Veterinärplatz 1, 1210 Vienna, Austria
M. Müller
Affiliation:
Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Veterinärplatz 1, 1210 Vienna, Austria
R. Claus*
Affiliation:
Fachgebiet Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung (470a), Universität Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany
*
†Corresponding author E-mail:[email protected]
Get access

Abstract

Glucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1 kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15 kDa protein which corresponds to the estimated molecular weight for the receptor fragment.

After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.

The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.

Keywords

amino acid sequencesglucocorticoidsimmunohistochemistrypigswestern blotting
Type
Growth, development and meat science
Information
Animal Science , Volume 73 , Issue 2 , October 2001 , pp. 267 - 278
Copyright
Copyright © British Society of Animal Science 2001

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Adams, B., Dörfler, P., Aguzzi, A., Kozmik, Z., Urbánek, P., Mawer-Fogg, I. and Busslinger, M. 1992. Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes and Development 6: 1589–1607.Google Scholar
Böck, P. 1989. Romeis Mikroscopische Technik. Urban and Schwarzenberg, Munich.Google Scholar
Cidlowski, J. A., Bellingham, D. L., Powell-Oliver, F.E, Lubahn, D. B. and Sar, M. 1990. Novel antipeptide antibodies to the human glucocorticoid receptor. Recognition of multiple forms in vitro and distinct localization of cytoplasmic and nuclear receptors. Molecular Endorinology 4: 1427–1437.Google Scholar
Claassen, E., Zegers, N.D., Laman, J. D. and Boersma, W. J. 1993. Use of synthetic peptides for the production of site (amino acid) specific polyclonal and monoclonal antibodies. The Year of Immunology 7: 150–161.Google Scholar
Claus, R., Raab, S. and Dehnhard, M. 1996. Glucocorticoid receptors in the pig intestinal tract and muscle tissues. Journal of Veterinary Medicine Series A 43: 553–560.Google Scholar
Danielson, M., Northrop, J. P. and Ringlold, G. M. 1986. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wildtype and mutant receptor proteins. EMBO Journal 5: 2513–2520.Google Scholar
Evans, R. M. 1988. The steroid and thyroid hormone receptor superfamily. Science 240: 889–895.Google Scholar
Fuller, P. J. 1991. The steroid receptor superfamily: mechanisms of diversity. FASEB Monographs 5: 3092–3099.CrossRefGoogle ScholarPubMed
Gao, X., Kalkhoven, E., Peterson-Maduro, J., Burg, B. van der and Destree, O. H. 1994. Expression of the glucocorticoid receptor gene is regulated during early embryogenesis of Xenopus laevis . Biochimica et Biophysica Acta 1218: 194–198.Google Scholar
Hollenberger, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G. and Evans, R. M. 1985. Primary structure and expression of functional human glucocorticoid receptor cDNA. Nature 318: 635–641.CrossRefGoogle Scholar
Keightley, M. C. and Fuller, P. J. 1994. Unique sequences in the guinea pig glucocorticoid receptor induce constitutive transactivation and decrease steroid sensitivity. Molecular Endocrinology 8: 431–439.Google Scholar
Klemcke, H. G. 2000. Isolation of the porcine glucocorticoid receptor cDNA. GenBank AY007222.Google Scholar
Kofler, R. 2000. The molecular basis of glucocorticoid-induced apoptosis of lymphoblastic leukemia cells. Histochemical and Cellular Biology 114: 1–7.Google Scholar
Miesfeld, R., Rusconi, S., Godowski, P., Maler, B. A., Okret, S., Wikstrom, A. C., Gustafsson, J. A. and Yamamoto, K. R. 1986. Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell 46: 389–399.Google Scholar
Miller, A. H., Spencer, R. L., Pearce, B. D., Pisell, T. L., Azrieli, Y., Tanapa, P., Moday, H., Rhee, R. and McEwan, B. S. 1998. Glucocorticoid receptors are differentially expressed in the cells and tissues of the immune system. Cellular Immunology 186: 45–54.CrossRefGoogle ScholarPubMed
Miller, A. H., Spencer, R. L., Stein, M. and McEven, B. S. 1990. Adrenal steroid receptor binding in spleen and thymus after stress or dexamethasone. American Journal of Physiology 259: E405–E412.Google Scholar
Moran, T. J., Gray, S., Mikosz, C. A. and Conzen, S.D. 2000. The glucocorticoid receptor mediates a survival signal in human mammary epithelial cells. Cancer Research 60: 867–872.Google Scholar
Oakley, R. H. and Cidlowski, J. A. 1993. Homologous down regulation of the glucocorticoid receptor: the molecular machinery. Critical Reviews in Eukaryotic Gene Expression 3: 63–88.Google ScholarPubMed
Perreau, V. and Moisan, M. P. 1997. Sus scrofa glucocorticoid receptor partial cDNA (hormone binding domain). Genbank Accession no. U88894.Google Scholar
Quaroni, A., Tian, J. Q., M., Göke and Podolsky, D. K. 1999. Glucocorticoids have pleiotropic effects on small intestinal crypt cells. American Journal of Physiology 277: G1027–G1040.Google ScholarPubMed
Reynolds, P. D., Pittler, S. J. and Scammel, J. G. 1997. Cloning and expression of the glucocorticoid receptor from the squirrel monkey (Saimiri boliviensis boliviensis), a glucocorticoid resistance primate. Journal of Clinical Endocrinology and Metabolism 82: 465–472.Google Scholar
Riccardi, C., Cifone, M. G. and Migliorati, G. 1999. Glucocorticoid hormone-induced modulation of gene expression and regulation of T-cell death: role of GITR and GILZ, two dexamethasone-induced genes. Cellular Death Differentiation 6: 1182–1189.Google Scholar
Sharif, S. and Mallard, B. A. 1995. Partial sequence of exon 2 of the cattle, sheep and pig glucocorticoid receptor gene, containing CAG repeats. Genbank Accession no. U37385.Google Scholar
Thompson, E. B. 1994. Apoptosis and steroid hormones. Molecular Endocrinology 8: 665–673.Google Scholar
Vanderbilt, J. N., Miesfeld, R., Maler, B. A. and Yamamoto, K. R. 1987. Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity. Molecular Endocrinology 1: 68–74.Google Scholar
Weaver, S. A., Schaefer, A. L. and Dixon, W. T. 2000. Western blotting for detection of glucocorticoid receptors in the brain and pituitary gland from adrenal intact pigs. Brain Research 869: 130–136.Google Scholar
Wilson, E. M. 1998. Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor. Molecular Endocrinology 2: 1018–1025.CrossRefGoogle Scholar
Yang, K., Hammond, G. L. and Challis, J. R. 1992. characterization of an ovine glucocorticoid receptor cDNA and developmental changes in mRNA levels in fetal sheep hypothalamus, pituitary gland and adrenal. Journal of Molecular Endocrinology 8: 173–180.CrossRefGoogle ScholarPubMed
Zegers, N. D., Claassen, E., Neelen, C., Mulder, E., Laar, J. H. van, Voorhorst, M. M., Berrevoets, C. A., Brinkman, A. O., Kwast, T. H. van der and Ruizveld de Winter, J. A. 1991. Epitope prediction and conformation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy. Biochimica et Biophysica Acta 1073: 23–32.CrossRefGoogle ScholarPubMed