Liposome-mediated cellular delivery of active gp91phox

Abstract

Background. Gp91phox is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulamatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. Methodology. Here we optimize a prokaryatic cell-free expression system to produce integral mammalian membrane proteins. Conclusions. Using this system, we over-express truncated forms of the gp91phox 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 recombinant gp91phox proteins to the plasma membrane. Our data support the concept of 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 ptotein. © 2007 Marques et al.