α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression.

Cell-free protein synthesis is becoming a serious alternative to cell-based protein expression. Cell-free systems can deliver large amounts of cytoplasmic recombinant proteins after a few hours of incubation. Recent studies have shown that membrane proteins can be also expressed in cell-free reactions and directly inserted into phospholipid membranes. In this work, we present a quantitative method to study in real time the concurrent cell-free expression and insertion of membrane proteins into phospholipid bilayers. The pore-forming protein α-hemolysin, fused to the reporter protein eGFP, was used as a model of membrane protein. Cell-free expression of the toxin in solution and inside large synthetic phospholipid vesicles was measured by fluorometry and fluorescence microscopy respectively. A quartz crystal microbalance with dissipation was used to characterize the interaction of the protein with a supported phospholipid bilayer. The cell-free reaction was directly incubated onto the bilayer inside the microbalance chamber while the frequency and the dissipation signals were monitored. The presence of pores in the phospholipid bilayer was confirmed by atomic force microscopy. A model is presented which describes the kinetics of adsorption of the expressed protein on the phospholipid bilayer. The combination of cell-free expression, fluorescence microscopy and quartz crystal microbalance-dissipation is a new quantitative approach to study the interaction of membrane proteins with phospholipid bilayers.