Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

Abstract

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.