Expression, purification, and structural prediction of the Ets transcription factor ERM.
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 alpha-helix. In contrast, the 250-residue central domain seems to have very little structure.
Mauen
Sébastien
S
Laboratoire de Virologie Moléculaire-Faculté de Médecine, ULB, CP 614, 808 route de Lennik, 1070 Brussels, Belgium.
Huvent
Isabelle
I
Raussens
Vincent
V
Demonte
Dominique
D
Baert
Jean-Luc
JL
Tricot
Catherine
C
Ruysschaert
Jean-Marie
JM
Van Lint
Carine
C
Moguilevsky
Nicole
N
de Launoit
Yvan
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors