Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system.

Abstract Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b 558 (Cyt b 558 ) which consists of a heterodimer of the gp91 phox and p22 phox subunits. The Cyt b 558 is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cyt b 558 (rCyt b 558 ). We expressed the gp91 phox and p22 phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91 phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCyt b 558 displayed comparable spectral properties to neutrophil Cyt b 558 . In contrast to rCyt b 558 produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein–protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.