Synthesis and biophysical characterization of aquaporin water channels produced in an Escherichia coli-based cell-free protein expression system

Cell-free protein expression technology is a robust and reliable method for producing proteins, offering many advantages over traditional in vivo synthesis. To date, the majority of proteins expressed in cell-free systems have been cytoplasmic in origin. In this work we show the feasibility of cell-free synthesis of a membrane protein, a class of proteins that have traditionally been very difficult to express.; As our models we selected the E. coli water channel AqpZ and human water transporter AQP4. We have shown that both AqpZ and AQP4 express and insert into synthetic vesicles in the E. coli-based cell-free combined transcription and translation protein expression system. Our total yields were ∼650 mug/ml AqpZ and ∼600 mug/ml (AQP4), and approximately 40% AqpZ and 26% (AQP4) of that was recovered in the synthetic vesicles. AqpZ is functional and resistant to protease degradation, suggesting that it is properly inserted. AQP4, on the other hand, did not exhibit any functional activity and was susceptible to protease degradation. We also observed that for both, AqpZ and AQP4, the amount of protein inserted into the membrane is independent of the surface area available for insertion, suggesting that the limiting factor is a component of the cell-free extract.; In order to express and recover membrane proteins from the cell-free reaction, we developed a method for chromatographic purification of synthetic vesicles. This technique is based on the reversible binding of biotin to monomeric avidin. Biotin-functionalized lipids were integrated into synthetic vesicles and the vesicles were recovered on a monomeric avidin column. We thoroughly characterized this technique and modeled it using Langmuir adsorption theory.; We employed stopped-flow static light scattering, Black Lipid Membranes (BLM), and Quartz Crystal Microbalance (QCM) to functionally assay AqpZ. BLM and QCM both confirmed functional activity qualitatively, and we were able to obtain specific activity via stopped-flow static light scattering. Our measured functional activity for AqpZ falls within the range of previously reported values. Additionally, we developed a simple technique for lipid transition phase temperature (Tm) determination at very low vesicle concentrations using stopped-flow static light scattering.