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1996 | 43 | 4 |

Tytuł artykułu

Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from Drosophila melanogaster

Warianty tytułu

Języki publikacji

EN

Abstrakty

EN
Two members of the nuclear receptor superfamily, EcR and UltTaspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone — the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.

Wydawca

-

Rocznik

Tom

43

Numer

4

Opis fizyczny

p.611-621,fig.

Twórcy

autor
  • Technical University of Wroclaw, Wybrzeze S.Wyspianskiego 27, 50-370 Wroclaw, Poland
autor
autor

Bibliografia

  • 1. Gronemeyer, H. & Laudet, V. (1995) Transcrip­tion factors 3: Nuclear receptors; in Protein Pro­file (Sheterline, P., ed.) vol. 2, pp. 1173-1308, Academic Press.
  • 2. Evans, R.M. (1988) The steroid and thyroid hor­mone receptor super family. Science 240, 889- -895.
  • 3. Berg, J.M. (1989) DNA binding specificity of steroid receptors. Cell 57,1065-1068.
  • 4. Glass, C.K. (1994) Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocrine Rev. 15, 391-407.
  • 5. Truss, M. & Beato, M. (1993) Steroid hormone receptors: interaction with DNA and transcrip­tion factors. Endocrine Rev. 14,459-479.
  • 6. Mangelsdorf, D.J. & Evans, R.M. (1995)The RXR heterodimers and orphan receptors. Cell 83, 841-850.
  • 7. Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, P. & Hogness, D.S. (1991) The Drosophila EcR gene encodes an ecdysone rcceptor, a new member of the steroid receptor superfamily. Cell 67, 59-77.
  • 8. Henrich, V.C., Sliter, T.J., Lubahn, D.B., Maclntyre, A. & Gilbert, L.I. (1990) A steroid/ /thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue. Nucleic Acids Res. 18, 4143-4148.
  • 9. Yao, T.-P., Segraves, W.A., Oro, A.E., McKeown, M. & Evans, R.M. (1992) Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71, 63-72.
  • 10. Thomas, H.E., Stunnenberg, H.G. & Stewart, A.F. (1993) Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature (London) 362,471-475.
  • 11. Riddiford, L.M. (1993) Hormones and Droso­phila development; in The Development of Dro­sophila melanogaster (Bate, M. & Martinez Arias, A., eds.) pp. 899-939, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • 12. Riddihough, G. & Pelham, H.R.B. (1987) An ecdysone response element in the Drosophila hsp 27 promoter. EMBO J. 6,3729-3734.
  • 13. Ożyhar, A., Strangmann-Diekmann, M., Kiltz, H.-H. & Pongs, O. (1991) Characterisation of a specific ecdysteroid-DNA complex reveals common properties for invertebrate and ver­tebrate hormone-receptor/DNA interactions. Eur. J. Biochem. 200,329-335.
  • 14. Gallanger, S.R. (1987) in Current Protocols in Mol­ecular Biology (Asubel, P.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K., eds.) pp. A.3.9. — A.3.8., Greene Pub­lishing Associates and Wiley-Interscience, New York.
  • 15. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning. A Laboratory Manual. (Ford, N., Nolan, C. & Ferguson, M., eds.), Cold Spring Harbor Laboratory Press, Cold Spring I larbor. New York.
  • 16. Fried, M. & Crothers, D.M. (1981) Equilibria and kinetics of lac repressor-operator interactions by Polyacrylamide gel electrophoresis. Nucleic Acids Res. 9,6505-6525.
  • 17. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685.
  • 18. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72,248-254.
  • 19. Nardulli, A.M., Lew, D., Erijman, L., Shapiro, D.J. (1991) Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-res­ponsive promoter in cultured cells. J. Biol. Chem. 35,24070-24076.
  • 20. Dahlman, K.,Strömstedt, P.-E., Rae, C., Jörn vail, H., Flock, J.-L, Garlstedt-Duke, J. & Gustafsson, J.-A. (1989) High level expression in Escherichia coli of the DNA-binding domain of the glucocorticoid receptor in a functional formutilizing domain specific cleavage of a fusion protein. /. Biol. Chem. 264, 80ł-809.
  • 21. Georgiou, G., Valax, P., Ostermeier, M. & Horowitz, P.M. (1994) Folding and aggregation of TEM ß-lactamase: Analogies with the formation of inclusion bodies in Escherichia coli. Protein Sei. 3,1953-1960.
  • 22. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. & Stüber, D. (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/ /Technology 6,1321-1325.
  • 23. Hockncy, R.C. (1994) Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 12,456-463.
  • 24. Georgiou, G. & Valax, P. (1996) Expression of correctly folded proteins in Escherichia coli. Curr. Opin. Biotechnol. 7,190-197.

Typ dokumentu

Bibliografia

Identyfikatory

Identyfikator YADDA

bwmeta1.element.agro-article-f84e4f7f-2243-4817-a1c8-ecb57f2d4590
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