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Title
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Expression and purification of a recombinant DNA-binding domain of ADR6 protein from Escherichia coli and its secondary structure characterization.
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Authors
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X.Tu,
Y.Xiao,
W.Zeng,
Y.Shi.
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Ref.
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Biochim Biophys Acta, 2000,
1481,
167-174.
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PubMed id
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Abstract
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From Saccharomyces cerevisiae, a piece of ADR6 gene that encodes a DNA-binding
domain of ADR6 protein was cloned and expressed in Escherichia coli. With
Ni-chelating column and high-performance liquid chromatography (HPLC), This
recombinant protein (RDB-ADR6) could reach more than 95% purity. The molecular
weight (MW) of RDB-ADR6 is 13405 Da with mass spectra technique containing 114
amino acid residues. Structural aspects of RDB-ADR6 were examined by
spectroscopic techniques. It contains approximately 25% alpha-helix and 24%
beta-turn both with circular dichroism (CD) and Fourier transform infrared
spectroscopy (FTIR). Percent of beta-sheet differs between these two methods in
that 22% in CD while 35% in FTIR. RDB-ADR6 contains only one tryptophan residue.
Fluorescence studies show that this residue may lie in a hydrophobic
circumstance either on or near the surface of the molecule. This was confirmed
by a blue shift of 20 nm in the fluorescence emission spectrum as compared to
the protein in 6 M guanidine hydrochloride (GuHCl) and by quenching studies with
KI. Effects of different pH and SDS in different concentration on the secondary
structure of RDB-ADR6 were also studied. A model was obtained by comparative
modeling with homologous known structure protein by program Modeller 4.
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