Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system.

Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from other sources. Photoactivable amino acid analogs used for p7 protein synthesis enabled oligomerization state analysis in liposomes by cross-linking. Therefore, these findings emphasize the quality of the cell-free produced p7 proteoliposomes which can benefit the field of the hepatitis C virus (HCV) protein production and characterization and also provide tools for the development of new inhibitors to reinforce our therapeutic arsenal against HCV.

Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems.

The mitochondrial voltage-dependent anion channel (VDAC) is a pivotal protein since it provides the major transport pathway between the cytosol and the mitochondrial intermembrane space and it is implicated in cell apoptosis by functioning as a gatekeeper for the trafficking of mitochondrial death molecules. VDAC is a beta-barrel channel with a large conductance, and we use it as a model transport protein for the design of biomimetic systems. To overcome the limitations of classical overexpression methods for producing and purifying membrane proteins (MPs) we describe here the use of an optimized cell-free system. In a one-step reaction VDAC is obtained directly integrated into liposomes and purified by ultracentrifugation. We then combine proteoliposomes with different bilayers models in order to validate VDAC insertion and functionality. This VDAC biomimetic model is the first example validating the use of a cell-free expression system for production of MPs into liposomes and tethered bilayers as a toolbox to build a wide range of biomimetic devices.

[Biomimetic sensors in biomedical research]. / Les capteurs biomimétiques en recherche biomédicale.

The recent research on both the synthesis of membrane proteins by cell-free systems and the reconstruction of planar lipid membranes, has led to the development of a cross-technology to produce biosensors or filters. Numerous biomimetic membranes are currently being standardized and used by the industry, such as filters containing aquaporin for water desalination, or used in routine at the laboratory scale, for example the bacteriorhodopsin as a light sensor. In the medical area, several fields of application of these biomimetic membranes are under consideration today, particularly for the screening of therapeutic molecules and for the developing of new tools in diagnosis, patient monitoring and personalized medicine.

Anti-Tumor Effects of Bak-Proteoliposomes against Glioblastoma.

Despite palliative treatments, glioblastoma (GBM) remains a devastating malignancy with a mean survival of about 15 months after diagnosis. Programmed cell-death is de-regulated in almost all GBM and the re-activation of the mitochondrial apoptotic pathway through exogenous bioactive proteins may represent a powerful therapeutic tool to treat multidrug resistant GBM. We have reported that human Bak protein integrated in Liposomes (LB) was able, in vitro, to activate the mitochondrial apoptotic pathway in colon cancer cells. To evaluate the anti-tumor effects of LB on GBM, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and Western blot analysis were performed on GL26 murine cell line. LB treatment shows a dose-dependent inhibition of cell viability, followed by an up-regulation of Bax and a down-modulation of JNK1 proteins. In GL26-bearing mice, two different routes of administration were tested: intra-tumor and intravenous. Biodistribution, tumor growth and animal survival rates were followed. LB show long-lasting tumor accumulation. Moreover, the intra-tumor administration of LB induces tumor growth delay and total tumor regression in about 40% of treated mice, while the intravenous injection leads to a significant increased life span of mice paralleled by an increased tumor cells apoptosis. Our findings are functional to the design of LB with potentiated therapeutic efficacy for GBM.

Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes.

Pseudomonas aeruginosa is the second-leading cause of nosocomial infections and pneumonia in hospitals. Because of its extraordinary capacity for developing resistance to antibiotics, treating infections by Pseudomonas is becoming a challenge, lengthening hospital stays, and increasing medical costs and mortality. The outer membrane protein OprF is a well-conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. Here, we used a bacterial cell-free expression system to reconstitute OprF under its native forms in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa Remarkably, we showed that our system promotes the folding of OprF into its active open oligomerized state as well as the formation of mega-pores. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.

Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins.

The delivery of functional therapeutic proteins by lipid vesicles into targeted living cells is one of the most promising strategies for treatment of different diseases and cancer. The use of this system in the delivery of membrane proteins directly into cells remains to be tested because the methods for producing membrane proteins are difficult to perform. Here we describe the effect of proteoliposomes containing the voltage-dependent anion channel (VDAC) and pro-apoptotic Bak, both produced with an optimized cell-free expression system. For the first time, recombinant VDAC and Bak proteins are synthesized and directly integrated into the lipidic bilayer of natural liposomes in a one-step reaction. VDAC has been shown to play an essential role in apoptosis in mammalian cells by regulating cytochrome c release from mitochondria and Bak modulates mitochondrial membrane permeability upon activation. Internalization of recombinant proteoliposomes into mammalian cells induces apoptosis by release of cytochrome c and caspases activation. These results highlight that membrane proteins integrated in natural liposomes can represent an excellent candidate for cancer protein therapy.