Very high membrane density of heterologously expressed human Aquaporin 1. It is approximately fourteen times higher than previously described [32] and to our knowledge the membrane density obtained in this study is unprecedented for a recombinantly produced human membrane protein. The potential of the present expression system for structure-function and structural studies of mammalian membrane proteins is obvious when compared to the densities of recombinant membrane protein previously serving as starting point for purification and crystallization of mammalian membrane proteins. The densities obtained for 7TM receptors were all in the range of 50 pmol/mg total membrane protein (corresponding to 0.2 of total membrane protein content) after expression in P. pastoris orFigure 4. Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes. A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15uC at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose. doi:10.1371/journal.pone.0056431.gHigh Level Human Aquaporin Production in YeastFigure 5. Endo glycosidase H treatment of yeast crude membranes. 4, crude membranes from yeast producing hAQP1GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His. doi:10.1371/journal.pone.0056431.ginsect cells [6,45]. The expression system described here is able to deliver around 1,500 pmol hAQP1-GFP/mg total membrane protein corresponding to 8.5 of total membrane protein content. A high membrane density in the starting material is a significant advantage for purification of large amounts of recombinant membrane protein. Using our previously described fermentation setup [34] the present expression system may deliver in the range of 350 mg human Aquaporin-1 per 200 grams of yeast cells after a single fermentation. To ease quantification of correctly folded hAQP1, sub-cellular localization, in vivo folding efficiency and development of a purification protocol, we chose to produce hAQP1 C-terminally tagged with GFP and an eight histidines long purification 
tag, a concept previously described [46]. The expression system described in the present paper extends work initially developed for recombinant production of the a1,b1 pig kidney Na,K-ATPase [34]. In agreement with these results the combined use of a galactose inducible promoter, an expression plasmid with an adjustable copy AZ 876 web number, a protease deficient yeast host strainFigure 7. Detergent screen for solubilization of hAQP1-GFP8His. Crude membranes were solubilized as described in Materials and Methods. GFP fluorescence was used to calculate percent solubilized hAQP1-GFP-8His. Abbreviations used; DM, n-decyl-b-D-maltopyranoside; DDM, n-dodecyl-b -D-maltopyranoside; OG, TA02 price n-octyl-b -D-glucopyranoside; CHAPS, 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate/N,N-Dimethyl-3-Sulfo-N-[3-[[3a,5b,7a,12a)-3,7,12-Trihydroxy24-Oxocholan-24-yl]Amino]propyl]-1-Propanaminium Hydroxide; CYMAL-5, 5-Cyclohexyl-1-Pentyl-b-D-Maltoside; Fos-12, n-Dodecylphosphocholine. doi:10.1371/journal.pone.0056431.gFigure 6. Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1. (A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence.Very high membrane 
density of heterologously expressed human Aquaporin 1. It is approximately fourteen times higher than previously described [32] and to our knowledge the membrane density obtained in this study is unprecedented for a recombinantly produced human membrane protein. The potential of the present expression system for structure-function and structural studies of mammalian membrane proteins is obvious when compared to the densities of recombinant membrane protein previously serving as starting point for purification and crystallization of mammalian membrane proteins. The densities obtained for 7TM receptors were all in the range of 50 pmol/mg total membrane protein (corresponding to 0.2 of total membrane protein content) after expression in P. pastoris orFigure 4. Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes. A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15uC at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose. doi:10.1371/journal.pone.0056431.gHigh Level Human Aquaporin Production in YeastFigure 5. Endo glycosidase H treatment of yeast crude membranes. 4, crude membranes from yeast producing hAQP1GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His. doi:10.1371/journal.pone.0056431.ginsect cells [6,45]. The expression system described here is able to deliver around 1,500 pmol hAQP1-GFP/mg total membrane protein corresponding to 8.5 of total membrane protein content. A high membrane density in the starting material is a significant advantage for purification of large amounts of recombinant membrane protein. Using our previously described fermentation setup [34] the present expression system may deliver in the range of 350 mg human Aquaporin-1 per 200 grams of yeast cells after a single fermentation. To ease quantification of correctly folded hAQP1, sub-cellular localization, in vivo folding efficiency and development of a purification protocol, we chose to produce hAQP1 C-terminally tagged with GFP and an eight histidines long purification tag, a concept previously described [46]. The expression system described in the present paper extends work initially developed for recombinant production of the a1,b1 pig kidney Na,K-ATPase [34]. In agreement with these results the combined use of a galactose inducible promoter, an expression plasmid with an adjustable copy number, a protease deficient yeast host strainFigure 7. Detergent screen for solubilization of hAQP1-GFP8His. Crude membranes were solubilized as described in Materials and Methods. GFP fluorescence was used to calculate percent solubilized hAQP1-GFP-8His. Abbreviations used; DM, n-decyl-b-D-maltopyranoside; DDM, n-dodecyl-b -D-maltopyranoside; OG, n-octyl-b -D-glucopyranoside; CHAPS, 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate/N,N-Dimethyl-3-Sulfo-N-[3-[[3a,5b,7a,12a)-3,7,12-Trihydroxy24-Oxocholan-24-yl]Amino]propyl]-1-Propanaminium Hydroxide; CYMAL-5, 5-Cyclohexyl-1-Pentyl-b-D-Maltoside; Fos-12, n-Dodecylphosphocholine. doi:10.1371/journal.pone.0056431.gFigure 6. Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1. (A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence.