Expression, purification, and structural prediction of the Ets transcription factor ERM.

S Mauen, I Huvent, V Raussens, D Demonte, JL Baert, C Tricot, JM Ruysschaert, C Van Lint, Nicole Moguilevsky, Y de Launoit

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    Résumé

    The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors.Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an α-helix. In contrast, the 250-residue central domain seems to have very little structure.
    langue originaleAnglais
    Pages (de - à)1192-1201
    Nombre de pages10
    journalBiochimica et Biophysica Acta - General Subjects
    Volume1760
    Numéro de publication8
    Les DOIs
    Etat de la publicationPublié - 2006

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