Figure 2.

Optimized production of NOX2/p22^phox liposomes using the cell-free expression system.

Notes: (A) Effect of magnesium and potassium concentrations on cell-free production of p22^phox protein. In vitro expression was performed in 96-well plates with a volume of 50 µL. (B) Effect of lipid composition on the expression of p22^phox subunit. Total protein fraction was obtained after the cell-free expression reaction, while the proteoliposome fraction was separated after a discontinuous sucrose gradient to separate them from liposomes and aggregated proteins. (C) Effect of the reaction time variation on NOX2 and p22^phox expression. Reactions were carried out at 30°C for 2 h, 4 h, 6 h or 16 h, in batch format (100 µL) and separated by SDS-PAGE on a 15% gel. P22^phox and NOX2 detection bands are indicated with stars. (D) Effect of the variation of iron and hemin concentration on protein expression. In vitro expression was performed in 96-well plates with a volume of 50 µL. In all experiments, monoclonal anti-His HRP-conjugated antibody and anti-NOX2 antibodies (clone 44.1) were used for the detection of p22^phox and NOX2, respectively. Optimal concentrations were indicated. * Indicates the location of NOX2 and p22^phox.

Abbreviations: NTPs, nucleotide triphosphates; PLs, proteoliposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.