Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) 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. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) 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 (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) 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 gp91(phox) protein

DBD as a post-discharge bipolar ions source and selective ioninduced nucleation versus ions polarity

International audienceIons densities and mobilities in post-dielectric barrier discharge (post-DBD) are presented here. To extract ions from DBD and perform post-discharge measurements the best functioning conditions are low overpressure, high frequency (> 25 kHz, to avoid electro-collection) and low flow rate (1 lpm, to reduce dilution). Besides, ions densities in post-discharge increase with electrode temperature and, at low flow rates, with the number of discharge filaments by time and surface unit (controlled by voltage at fixed frequency). In both cases, the reinforcement of dielectric material surface polarization reduces the local electro-collection of ions inside DBD or increases the production of ions by subcritical avalanches outside filaments. Concerning mobility measurements, it is shown that for low saturation, vapours emitted from post-DBD polymer tubes only affect positive ions mobility due to selective ion-induced nucleation on positive ions. When metal post-DBD tubes are used, positive ions keep the same range of electric mobility at any temperature while there is a drop in negative ions mobility around 100 °C, probably related to a chemical transition between O3 and NOx

Functional expression of plant membrane proteins in Lactococcus lactis.

International audienceThe study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization

Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae

The Ca(2+)-ATPase SERCA1a (sarcoplasmic–endoplasmic reticulum Ca(2+)-ATPase isoform 1a) from rabbit has been overexpressed in Saccharomyces cerevisiae. This membrane protein was purified by avidin agarose affinity chromatography based on natural biotinylation in the expression host, followed by HPLC gel filtration. Both the functional and structural properties of the overexpressed protein validate the method. Thus, calcium-dependent ATPase activity and calcium transport are essentially intact after reconstitution in proteoliposomes. Moreover, the recombinant protein crystallizes in a form that is isomorphous to the native SERCA1a protein from rabbit, and the diffraction properties are similar. This represents a successful crystallization of a mammalian membrane protein derived from a heterologous expression system, and it opens the way for the study of mutant forms of SERCA1a