Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system.
Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b(558) (Cytb(558)) which consists of a heterodimer of the gp91(phox) and p22(phox) subunits. The Cytb(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 Cytb(558) (rCytb(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 rCytb(558) displayed comparable spectral properties to neutrophil Cytb(558). In contrast to rCytb(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.
Ostuni
Mariano A
MA
Laboratoire de Chimie-Physique, UMR8000, CNRS-Université Paris-Sud, 91405 Orsay, France.
Lamanuzzi
Leila B
LB
Bizouarn
Tania
T
Dagher
Marie-Claire
MC
Baciou
Laura
L
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003573:Cytochrome b Group / Q000096:biosynthesis*; D015870:Gene Expression; D006801:Humans; D007313:Insecta; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008563:Membrane Lipids / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis*; D010100:Oxygen / Q000378:metabolism; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013481:Superoxides / Q000378:metabolism
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D008563:Membrane Lipids; D011994:Recombinant Proteins; D013481:Superoxides; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D010100:Oxygen