Production of active truncated gp91^phox proteins

Introduction

GP91^phox is a membrane protein constituted of 6 transmembrane domains and the catalytic core of the NADPH oxidase complex of netrophils. A lack of this protein causes chronic granulomatous disease, a rare genetic disorder characterized by severe and recurrent infectious due to the incapacity of phagocytoses to kill microorganisms.

Methods

Different truncated versions of gp91^phox protein were produced either in a soluble form, using detergents, or directely integrated into the lipid bilayer of liposomes forming proteoliposomes. In order to evaluate the ability of recombinant proteoliposomes to deliver active gp91^phox truncated protein, Heal cells were incubated for 8 hours either with proteoliposomes or with empty liposomes or with soluble truncated gp91^phox.

Results

All recombinant proteins exhibit a specific enzymatic activity as show in figure 1 for the solubilised recombinant proteins and in figure 2 for the proteoliposome.

Figure 1. Diaphorase activity measurement of soluble truncated gp91^phox.

Different truncated version of gp91phox containing up to 2 transmembrane domains (TM) were produced. Assays were performed in presence of cytosolic extract from human neutrophils using two different electrons acceptors NBT (NitroBlue Tetrazolium) and INT (IodoNitro Tetrazolium).

Figure 2. Diaphorase activity measurement of truncated gp91^phox proteoliposome.

A truncated version of gp91^phox containing 4 transmembrane domains was overexpressed. Evaluation of the diaphorase activity was performed in presence or in absence of cytosolic extract from human neutrophils (respectively plus or minus activation) using two different electron acceptors NBT and INT.

Using confocal microscopy, the direct delivery of active gp91^phox recombinant protein into the plasma membrane of mammalian cells was demonstrated (figure 3).

Figure 3. Confocal analysis of the distribution of recombinant gp91^phox protein delivery with proteoliposomes in Hela cells after 8 hours of incubation.

As a negative control, cells were incubated with 0.5mg of empty liposomes or 0.5mg of soluble gp91^phox protein. Cells were labelled for the exogenous protein using an anti-gp91^phox antibody (Red) and for the endogenous Clathrin (Green).

Conclusion

Our data supports the concept of a cell-free expression technology for the production of active recombinant membrane proteins and their use as molecular tools for functional and structural studies or protein therapy. This cell free expression system demonstrates a rapid cellular delivery and relocated of the recombinant protein when expressed as proteoliposomes.