Overexpression and characterization of the Rhodobacter sphaeroides PufX membrane protein in Escherichia coli.
Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had alpha-helix contents of 29% in organic solvents and 22-26% in 0.8-2.0% (w/v) n-octyl beta-D-glucopyranoside solutions, suggesting that the PufX contains a substantial alpha-helical region composed of 18-22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 alpha- and beta-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system.
Onodera
Shiho
S
Department of Biomolecular Engineering, Tohoku University, Sendai, Japan.
Suzuki
Hiroaki
H
Shimada
Yuichiro
Y
Kobayashi
Masayuki
M
Nozawa
Tsunenori
T
Wang
Zheng-Yu
ZY
D000596:Amino Acids / Q000235:genetics*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D025541:Bacteriochlorophyll A / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005960:Glucosides / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D045342:Light-Harvesting Protein Complexes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; C081521:B800-850 light-harvesting complex, bacteria; D001426:Bacterial Proteins; D025541:Bacteriochlorophyll A; D005960:Glucosides; D045342:Light-Harvesting Protein Complexes; C088749:PufX protein, Rhodobacter; D012997:Solvents; C094744:light-harvesting complex 1, Rhodospirillum; C018619:octyl-beta-D-glucoside; D006639:Histidine