OVEREXPRESSION AND PURIFICATION OF CYTOCHROME-C-OXIDASE FROM RHODOBACTER-SPHAEROIDES

The aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides has bee n overexpressed up to seven fold over that in wild-type strains by eng ineering a multicopy plasmid with all the required oxidase genes and b y establishing optimum growth conditions. The two operons containing t he three structural genes and two assembly genes for cytochrome c oxid ase were ligated into a pUC19 vector and reintroduced into several oxi dase-deleted R. sphaeroides strains. Under conditions of relatively hi gh 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. spha eroides. An improved cytochrome c oxidase purification protocol is rep orted, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and p urity than previously obtained. (C)1998 Academic Press.