Overexpression and purification of cytochrome c oxidase from Rhodobacter sphaeroides.
The aa3-type cytochrome c oxidase of Rhodobacter sphaeroides has been overexpressed up to seven fold over that in wild-type strains by engineering a multicopy plasmid with all the required oxidase genes and by establishing optimum growth conditions. The two operons containing the three structural genes and two assembly genes for cytochrome c oxidase were ligated into a pUC19 vector and reintroduced into several oxidase-deleted R. sphaeroides strains. Under conditions of relatively high pH and maximal aeration, high levels of expression were observed. A smaller expression vector, pBBR1MCS, and a fructose promoter (fruP)5 were found not to enhance cytochrome c oxidase expression in R. sphaeroides. An improved cytochrome c oxidase purification protocol is reported, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and purity than previously obtained.
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824-1319, USA.
Y
Y
Zhen
J
J
Qian
K
K
Follmann
T
T
Hayward
T
T
Nilsson
M
M
Dahn
Y
Y
Hilmi
A G
AG
Hamer
J P
JP
Hosler
S
S
Ferguson-Miller
D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000302:isolation & purification; D005632:Fructose / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D005632:Fructose; D003576:Electron Transport Complex IV