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

Electrical, Thermal and Optical Diagnostics of an Atmospheric Plasma Jet System

Plasma diagnostics of atmospheric plasmas is a key tool in helping to understand processing performance issues. This paper presents an electrical, optical and thermographic imaging study of the PlasmaStream atmospheric plasma jet system. The system was found to exhibit three operating modes; one constricted/localized plasma and two extended volume plasmas. At low power and helium flows the plasma is localized at the electrodes and has the electrical properties of a corona/filamentary discharge with electrical chaotic temporal structure. With increasing discharge power and helium flow the plasma expands into the volume of the tube, becoming regular and homogeneous in appearance. Emission spectra show evidence of atomic oxygen, nitric oxide and the hydroxyl radical production. Plasma activated gas temperature deduced from the rotational temperature of nitrogen molecules was found to be of order of 400 K: whereas thermographic imaging of the quartz tube yielded surface temperatures between 319 and 347 K.<br/

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

Investigation of a scalable barrel atmospheric plasma reactor for the treatment of polymer particles

This study reports on the performance of a scalable barrel atmospheric plasma system for the treatment of polymer particles. A novel feature of the barrel system design is the use of a biased electrode, which also acts as the roller for the glass barrel. The plasma is generated using either helium or helium / oxygen gas mixtures. The reactor was used to activate 20 g batches of silicone, polypropylene (PP), acrylonitrile butadiene styrene (ABS) and polyethylene terephthalate (PET) particles, each with diameters in the range 3 to 5 mm. The effect of plasma treatment time and gas flow rate on the water contact angle of the treated polymer particles was examined. The polymer water contact angles decreased from up to 140° to less than 10° after the barrel plasma treatment (polymer dependent). X-ray photoelectron spectroscopy (XPS) analysis is used to monitor the effect of the plasma treatment on both PET and silicone polymer particles. Optical emission spectroscopy (OES) was used as a diagnostic tool to monitor changes in atomic and molecular species intensity with experimental conditions. Emission lines of helium, oxygen and molecular bands of OH, N2 and N2+ were monitored and correlated with their spatial distribution within the plasma chamber. Electrical characterisation studies demonstrated an increase in plasma power with increasing input voltage and helium flow rate. The heating effect of the plasma was monitored using an infrared thermographic camera, the maximum barrel temperature after 30 minutes treatment found to be 29°C. While the current barrel plasma system design can treat 20 g of polymer the system design has the potential to be readily scalable for the activation of larger batches of particles