Expression and purification of a recombinant DNA-binding domain of ADR6 protein from Escherichia coli and its secondary structure characterization.
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.
Tu
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X
Laboratory of Structure Biology, School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
Xiao
Y
Y
Zeng
W
W
Shi
Y
Y
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
D004268:DNA-Binding Proteins