Cell-Free Synthesis of Functional Aquaporin Z in Synthetic Liposomes

The challenges involved in producing sufficient quantities of aquaporins for precise biophysical characterization have limited our knowledge of this important class of molecules. This article describes a cell-free protein synthesis method for producing high concentrations of the E. coli water transporter, aquaporin Z (AqpZ), in synthetic liposomes. To our knowledge, this is the first report of in vitro synthesis of a membrane protein directly into synthetic liposomes with verified function, (i.e., transport activity and selectivity). Titration of DOPC lipid vesicles added to the cell-free reaction show that production yields of active AqpZ are dependent oil the concentration of DOPC lipid vesicles added to the cell-free reaction, with 224 +/- 24 lipids required per aquaporin monomer. Supplementation of the signal recognition particle receptor (FtsY) to the cell-free reaction increases production of vesicle-associated AqpZ but not active AqpZ. Cell-free reactions using 7 mg/mL lipids that were not supplemented with FtsY produced 507 +/- 11 mu g/mL of vesicle-associated AqpZ that exhibited a specific water transport activity of (2.2 +/- 0.3) x 10(-14) cm(3) s(-1) monomer(-1). Proteinase K protection, activation energy determination, and selectivity against glycerol and urea transport also confirmed the production of correctly folded AqpZ. This technique is capable of producing milligram quantities of aquaporin that can be readily assayed for function, facilitating biophysical characterization and high-throughput analysis.