Purification and characterization of the recombinant Na+-translocating NADH: quinone oxidoreductase from Vibrio cholerae
2002
The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), was cloned under the regulation of the PBAD promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, t...
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
8
References
97
Citations
NaN
KQI