In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of\r\nhuman Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an\r\nadjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of\r\nfunctional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of\r\na purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression\r\nat 15uC in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal\r\nmedium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded\r\nrecombinant Aquaporin-1 at 30uC was due to in vivo mal-folding. Reduction of the expression temperature to 15uC almost\r\ncompletely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1\r\naccumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in\r\nsolubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity\r\nchromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae\r\nwas not N-glycosylated in contrast to the protein found in human erythrocytes.
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