WP1: Fabrication of ion channel based RED membranes
The main hypothesis for the RED membrane design is that we identify and use suitable ion selective channels. We have selected mainly microbial channels which are well characterized, with no (or little) voltage-dependent gating and microbial channels are simpler and more robust than their eukaryotic counterparts. Based on the large scientific literature on ion channels we have selected the following Cl-, K+ and Na+ channels for the design of biomimetic anion and cation selective RED membranes: the microbial GEF1 and YadQ Cl- channels from S. cerevisiae and E. coli, respectively, the KscA and Kir2 inward rectifier K+ channel from S. levidans and man in addition to the NaChBac Na+ channel from B. halodurans.
The production and purification of tagged ion channel-GFP fusion proteins will use the method developed by us to produce proteins in large quantities. After construction of expression plasmids, host cells are cultured in 15 liter computer controlled fermenters to a high cell density. Subcellular localization is determined via fluorescence microscopy. Cytosol or membranes are purified and expression levels are estimated by measurements of GFP fluorescence. Channel proteins are purified by HPLC affinity chromatography and purity is evaluated by in-gel fluorescence and Coomassie stain of GFP tagged proteins. Cation and anion selective membranes for RED will be fabricated using purified ion channel proteins reconstituted in lipids/polymers suitable for stabilization in a membrane support material Akin to the approach we developed for aquaporin membranes.