Expression, purification and characterization of a high potential iron-sulfur protein from Acidithiobacillus ferrooxidans.
The high potential iron-sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe(4)S(4)] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron-sulfur cluster.
Zeng
Jia
J
Department of Bioengineering, School of Resources Processing and Bioengineering, Central South University, Changsha, 410083, P.R. China.
Jiang
Huidan
H
Liu
Yuandong
Y
Liu
Jianshe
J
Qiu
Guanzhou
G
D042763:Acidithiobacillus / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; C007943:high potential iron-sulfur protein; D003545:Cysteine