Due to the lipid nature of cellular membranes preventing transport of most solutes between the cytosol and the extracellular environment as well as from the cytosol to the interior of organelles, cellular homeostasis relies on integral membrane proteins allowing selective trans membrane movement of solutes. Aquaporins constitute a family of physiologically very important integral membrane proteins that are found in all three kingdoms, eubacteria, archaea and eukaryotes. As protein channels, they facilitate passive transport of water across cell membranes. In the present study the yeast Saccharomyces cerevisiae was exploited as a host for heterologous expression of human aquaporins. Aquaporin cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human aquaporin was C-terminally tagged with yeast-enhanced GFP to quantify functional expression, to determine sub-cellular localization, to estimate in vivo folding efficiency and to ease establishment of a purification protocol. We found functional human aquaporin-1 to constitute up to 8.5 percent of total membrane protein content after expression at 15oC in a yeast host over producing the Gal4p transcription factor and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30oC was due to in vivo mal-folding. Reduction of the expression temperature to 15oC almost completely prevented Aquaporin1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1.
Bomholt, J., Helix Nielsen, C., Scharff-Poulsen, P., & Pedersen, P. A. (2014). Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces Cerevisiae. BIOPHYSICAL JOURNAL, 106(2, Supplement 1), 557A-558A. https://doi.org/10.1016/j.bpj.2013.11.3099