The flavocytochrome b(558) (Cytb(558)), heterodimer of two membrane proteins gp91(phox) and p22(phox), 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. Cytb(558) 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 p22(phox)-termini. Expressed alone with N-terminus fusion-tag, a production of stable and mature p22(phox) 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 p22(phox) was not stably expressed and was likely shuttled elsewhere to avoid any troubles of the biomass. The stability of the C-terminus tagged p22(phox) was restored when the protein was co-expressed with gp91(phox). Our data support the idea that Pichia pastoris presents the criteria of tangible cell factory for the production of the recombinant membrane Cytb(558) 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. (C) 2016 Elsevier Ltd. All rights reserved.