Preparation of cationic proteoliposomes using cell-free membrane protein synthesis: the chaperoning effect of cationic liposomes

Membrane protein reconstituted cationic liposomes are constructed using cell-free membrane protein synthesis in the presence of cationic liposomes. The chaperon effect of cationic liposomal membrane assists in folding the functional conformation of membrane protein. This preparation method enables the provision of the usage of proteoliposomes for drug delivery.


Measurement of the interactions between cationic liposomes and ribosomes
The average size of liposomes and ribosomes were measured as described above.

Preparation and purification of membrane protein-integrated proteoliposomes
Membrane protein-integrated liposomes were prepared using PURESYSTEM (PUREfrex®1.0; GeneFrontier, Chiba, Japan) as described previously. [2] In brief, the reaction mixture containing 4 ng/μL pDNA for Cx43 or 3 ng/μL pDNA for Cx43-EGFP was incubated with or without the indicated concentration of lipids (liposomes) without agitation for 4 h at 37 °C in a heat block incubator. For purification, a 40 μL sample was overlaid with 40 μL of 15% (w/v) sucrose solution and ultracentrifuged at 163,000 × g at 4 °C for 2 h. The 60 μL upper layer was collected as the supernatant sample and the lower 20 μL fraction was collected as the pellet sample.

Evaluation of the amount of synthesized connexin-43 and its solubility
To evaluate the amount of synthesized Cx43 and Cx43-EGFP, western blot analysis was performed using a SNAP i.d. ® 2.0 Protein Detection System (Millipore, Billerica, MA, USA) as described previously. [2] In brief, after separation by SDS-PAGE, protein bands were transferred electrophoretically to a polyvinylidene difluoride membrane. After blocking with Blockingone (Nacali Tesque, Kyoto, Japan), the membrane was incubated with a mouse anti-connexin-43 monoclonal IgG (BD Transduction Laboratories™, Lexington, KY, USA), and then incubated with goat antimouse IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then the membrane was reacted with ECL Western Blotting Detection Reagent (GE Healthcare) and bands were visualized and quantified using ImageQuants™ LAS4000 (GE Healthcare).

Confocal laser scanning microscopy (CLSM) observations
After the cell-free Cx43 synthesis in the presence of 4 mM lipid (liposomes), the cell-free reactants were added to a solution of SYBR Gold (1:2,500 dilution; Molecular Probes™/Thermo Fisher Scientific, Eugene, OR, USA). After mixing gently, the image was captured using CLSM (LSM780; Carl Zeiss, Oberkochen, Germany) with an excitation wavelength of 488 nm for SYBR Gold.

Measurement of connexin-43-EGFP production
The fluorescence intensity of Cx43-EGFP was measured at an excitation wavelength of 480 nm and emission wavelength of 510 nm using a fluorescence spectrometer (FP-8500; JASCO, Tokyo, Japan) equipped with Peltier thermostat cell holder system (ETC-815; JASCO). The cell-free reaction mixture containing 3 ng/μL pURE-Cx43-EGFP was incubated with or without 4 mM lipid (liposomes) at 37 °C and the time-course of synthesized cell-free Cx43-EGFP was recorded by measuring the increase in fluorescence intensity.

Statistical analysis
Differences in the solubility of Cx43 using the liposomes containing difference ratios of DOEPC and the inhibitory effects of the DOEPC liposomes on cell-free Cx43 synthesis were statistically evaluated using one-way factorial ANOVA followed by Tukey's multiple comparisons test. An adjusted P < 0.05 was considered statistically significant.
All statistical analyses were performed using Graphpad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Results are expressed as the mean ± standard deviation (n = 3).