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Ezzine, Aymen; Souabni, Hager; Machillot, Paul; Bizouarn, Tania; Baciou, Laura
Process biochemistry (1991), September 2016, Letnik: 51, Številka: 9Journal Article
Free C-terminus of p22phox allows a proper folding but a toxic effect on the yeast growth(right) and an increase oxidase activity capacity (left). Display omitted •Free- or tagged-p22phox termini determine production of mature and active heterodimer.•Overexpression of mature and well-folded His-p22phox decreased yeast cell biomass.•Instability of N-terminus free p22phox-His is counterbalanced by co-expression of gp91phox.•Free C-terminus of p22phox contributes to efficient NADPH oxidase. The flavocytochrome b558 (Cytb558), heterodimer of two membrane proteins gp91phox and p22phox, corresponds to the catalytic part of the phagocyte NADPH-oxidase. This complex plays a crucial role of the innate immune system by generating superoxide anion, the precursor of toxic reactive oxygen species. Cytb558 lacks of precise structural knowledge due to an inefficient production of pure protein. Aiming to optimize its production level in Pichia Pastoris, we explored the impact of the His-tag location on the p22phox-termini. Expressed alone with N-terminus fusion-tag, a production of stable and mature p22phox proteins was observed at the plasma membrane which was associated to a decrease of the yeast biomass (membrane limitation). In contrast, the C-terminus tagged p22phox was not stably expressed and was likely shuttled elsewhere to avoid any troubles of the biomass. The stability of the C-terminus tagged p22phox was restored when the protein was co-expressed with gp91phox. Our data support the idea that Pichia pastoris presents the criteria of tangible cell factory for the production of the recombinant membrane Cytb558 but an investigation in details on the optimal introduction of the tag was a prerequisite to obtain efficient production of mature proteins and active NADPH-oxidase complexes.
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