Expression and purification of the DNA-binding domain of the human transcription factor E2F1.
The DNA-binding domain of the human transcription factor E2F1 was expressed in Escherichia coli. Through a single purification step using a Ni2+ column, 40-50 mg of the highly purified recombinant protein was obtained from 1 liter of bacterial culture. In addition, it was shown that the recombinant protein had higher stability and solubility under acidic conditions than at a neutral or alkaline pH. The gel shift assay showed that the recombinant E2F1 DNA-binding domain was active in binding a fragment containing E2F sites. Circular dichroism measurements revealed that the recombinant protein approximately contains 33% alpha-helix, 11% beta-sheet, 5% turn, and 51% random coil at pH 7.0, and there was no obvious change for the secondary structure of the recombinant protein between pH 4.0 and pH 9.0. A 3D model was obtained by comparative protein modeling with a homologous protein whose structure was known by program Modeller 4. With the program DSSP, the predicted secondary structure content of the model was consistent with the result of circular dichroism spectrum.
Zeng
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W
Laboratory of Structural Biology, School of Life Science, University of Science and Technology of China, Hefei, Anhui, 230027, People's Republic of China.
Zhang
X
X
Tu
X
X
Guang
S
S
Xiao
Y
Y
Shi
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009532:Nickel; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D010446:Peptide Fragments; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D009532:Nickel