Abstract
BackgroundGp91phox is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms.MethodologyHere we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins.ConclusionsUsing this system, we over-express truncated forms of the gp91phox protein under soluble form in the presence of detergents or lipids resulting in active proteins with a “native-like” conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67phox, p47phox, p40phox and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91phox protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91phox proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91phox protein.
Highlights
Gp91phox protein is the catalytic subunit of the NADPH oxidase complex in human neutrophils and is involved in the electron transfer from NADPH to molecular oxygen O2 [1]
Each construct contains a histidine tag located either at the N- or at the C-terminus part of the protein (Figure 2A). These truncated gp91phox proteins were first cloned into vectors dedicated to a prokaryotic expression system and synthesized in vitro by using a transcriptiontranslation system using an E. coli lysate
221-C with the control cytosol and was strongly and significantly decreased in presence of p67phox-deficient cytosol (,0.9 mol/ min/mol gp91phox 221-C, Figure 4B). These results suggested a direct interaction between the cytosolic factors (p47phox, p67phox, p40phox and Rac) and the recombinant soluble proteins, indicating an assembly of these factors and the truncated proteins as earlier described for the native cytochrome b558 during the activation of the NADPH oxidase complex [1]
Summary
Gp91phox protein is the catalytic subunit of the NADPH oxidase complex in human neutrophils and is involved in the electron transfer from NADPH to molecular oxygen O2 [1]. NADPH oxidase is a multicomponent enzyme complex composed of cytochrome b558 (gp91phox and p22phox) and cytosolic factors (p67phox, p47phox, p40phox, Rac and Rap1A) that translocate at the membrane surface from cytosol upon stimulation. From this transfer and through the assembly of the constituents, NADPH oxidase is activated [5]. Gp91phox is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91phox protein
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