Expression and purification of a recombinant DNA-binding domain of ADR6 protein from Escherichia coli and its secondary structure characterization

• Published in: Biochimica et biophysica acta 2000-08, Vol.1481 (1), p.167-174
• Main Authors: Tu, Xiaoming, Xiao, Yazhong, Zeng, Wanyong, Shi, Yunyu
• Format: Article
• Language: English
• Subjects: ADR6; Amino Acid Sequence; Circular Dichroism; Cloning, Molecular; Comparative modeling; Conserved Sequence; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; FTIR; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary; Purification; Saccharomyces cerevisiae - metabolism; Sequence Alignment; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(00)00095-9

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% α-helix and 24% β-turn both with circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). Percent of β-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.