Characterization of the bacterially expressed Drosophila engrailed homeodomain.
To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli. Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction. Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography. The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion. UV-CD spectra of the En-HD showed that it mainly consisted of alpha-helix. Based on one-dimensional 1H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50 degrees C and low pH. The low pH resistance of the protein was also demonstrated by UV-CD measurement. Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis.
Institute for Protein Research, Osaka University.
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Yamamoto
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Yee
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Shirakawa
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Kyogoku
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D015342:DNA Probes; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D007301:Insect Hormones; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA