Ging were grown and induced for Aquaporin-1-GFP production as described [38]. Briefly, having reached an OD450 = 1.0 cells were diluted in the same medium to OD450 = 0.05, grown to OD450 < 0.5 and stained with FM4-64 [41] and DAPI [42]. Fluorescence was visualized at 10006 magnification with a Nikon Eclipse E600 fluorescence microscope equipped with an Optronics Magnafire model S99802 camera attached.Results Development of a S. cerevisiae expression system for human Aquaporin-To investigate the capacity of 25033180 S. cerevisiae for production of hAQP1 we constructed the expression plasmid pPAP8230 shown in Figure 1. Transcription of hAQP1-GFP-8His cDNA in PAP1500 S. cerevisiae cells transformed with pPAP8230 is driven by a CYC-GAL promoter and enhanced by application of a yeast strain overproducing the Gal4p transcriptional activator [34]. To potentially increase hAQP1 production the copy number of the expression plasmid can furthermore be increased ten times by selection for 125-65-5 hydrochloride.html”>order Emixustat (hydrochloride) leucine autotrophy [44].Detergent solubilization of Aquaporin-1-GFPInitial solubilization screens were performed using the popular detergent kit (D399-POP) from Affymetrix. This kit contains the six detergents CYMAL-5, Fos-Choline-12, n-Dodecyl-b-D- Maltoside, n-Decyl-b-D-Maltoside, CHAPS and n-Octyl-b-D-pyranoside. Crude membranes were incubated for 1 hour at 4uC with gentle reversal at a protein concentration of 2 mg/ml and a detergent: protein ratio of three in 50 mM phosphate buffer pH 7.5 containing 10 mM imidazole, 500 mM NaCl, 1 mM PMSF, 1 mg/ml Leupeptin, 1 mg/ml Pepstatin and 1 mg/ml Chymostatin. Solubilized and non-solubilized protein were separated by ultracentrifugation at 100,0006 g at 4uC for 30 minutes. GFP fluorescence in the supernatant and the pellet was measured in white microplates (Nunc, Denmark) in a microplate reader (Fluoroskan Ascent, Thermo Scientific, USA) and used to quantify solubilization efficiency. Excitation in these experiments was at 485 nm and emission was at 520 nm.Recombinant hAQP1-GFP accumulation depends on temperature and induction timeThe expression studies were performed in presence of all amino acids except leucine and isoleucine as we previously described these conditions to be beneficial for recombinant membrane protein accumulation [34]. Whole cell hAQP1-GFP fluorescence was used to determine the kinetics of accumulation of functional hAQP1with respect to induction temperature. Figure 2 shows thatNi-affinity purification of recombinant human AquaporinCYMAL-5 solubilized Aquaporin-1 protein was diluted ten times in buffer A (50 mM phosphate and 500 mM NaCl pH 7.5) containing 10 mM imidazole and incubated overnight at 4uC with Ni-NTA Superflow (Qiagen, Germany). A CellThru Disposable Column (Clontech, USA) was packed with the Ni-NTA slurry. The column was washed with ten volumes of buffer A containing 10 mM imidazole and 0.75 mg/ml CYMAL-5, 30 volumes of buffer A with 30 mM imidazole and 0.75 mg/ml CYMAL-5, seven volumes of buffer A with 100 mM imidazole and 0.75 mg/ ml CYMAL-5. Protein was eluted with three volumes of buffer A containing 250 mM imidazole and 0.75 mg/ml CYMAL-5 and subsequently with three volumes of buffer A with 500 mM imidazole and 0.75 mg/ml CYMAL-5. Eluted AQP1-GFP-8His protein was collected in fractions of 200 ml. All buffers contained 1 mM PMSF, 1 mg/ml Pepstatin, 1 mg/ml Chymostatin and 1 mg/ml Leupeptin.Figure 1. Structural map of the Aquaporin-1 expression plasmid pPAP8230. Abbreviations used: CYC-GALP,.Ging were grown and induced for Aquaporin-1-GFP production as described [38]. Briefly, having reached an OD450 = 1.0 cells were diluted in the same medium to OD450 = 0.05, grown to OD450 < 0.5 and stained with FM4-64 [41] and DAPI [42]. Fluorescence was visualized at 10006 magnification with a Nikon Eclipse E600 fluorescence microscope equipped with an Optronics Magnafire model S99802 camera attached.Results Development of a S. cerevisiae expression system for human Aquaporin-To investigate the capacity of 25033180 S. cerevisiae for production of hAQP1 we constructed the expression plasmid pPAP8230 shown in Figure 1. Transcription of hAQP1-GFP-8His cDNA in PAP1500 S. cerevisiae cells transformed with pPAP8230 is driven by a CYC-GAL promoter and enhanced by application of a yeast strain overproducing the Gal4p transcriptional activator [34]. To potentially increase hAQP1 production the copy number of the expression plasmid can furthermore be increased ten times by selection for leucine autotrophy [44].Detergent solubilization of Aquaporin-1-GFPInitial solubilization screens were performed using the popular detergent kit (D399-POP) from Affymetrix. This kit contains the six detergents CYMAL-5, Fos-Choline-12, n-Dodecyl-b-D- Maltoside, n-Decyl-b-D-Maltoside, CHAPS and n-Octyl-b-D-pyranoside. Crude membranes were incubated for 1 hour at 4uC with gentle reversal at a protein concentration of 2 mg/ml and a detergent: protein ratio of three in 50 mM phosphate buffer pH 7.5 containing 10 mM imidazole, 500 mM NaCl, 1 mM PMSF, 1 mg/ml Leupeptin, 1 mg/ml Pepstatin and 1 mg/ml Chymostatin. Solubilized and non-solubilized protein were separated by ultracentrifugation at 100,0006 g at 4uC for 30 minutes. GFP fluorescence in the supernatant and the pellet was measured in white microplates (Nunc, Denmark) in a microplate reader (Fluoroskan Ascent, Thermo Scientific, USA) and used to quantify solubilization efficiency. Excitation in these experiments was at 485 nm and emission was at 520 nm.Recombinant hAQP1-GFP accumulation depends on temperature and induction timeThe expression studies were performed in presence of all amino acids except leucine and isoleucine as we previously described these conditions to be beneficial for recombinant membrane protein accumulation [34]. Whole cell hAQP1-GFP fluorescence was used to determine the kinetics of accumulation of functional hAQP1with respect to induction temperature. Figure 2 shows thatNi-affinity purification of recombinant human AquaporinCYMAL-5 solubilized Aquaporin-1 protein was diluted ten times in buffer A (50 mM phosphate and 500 mM NaCl pH 7.5) containing 10 mM imidazole and incubated overnight at 4uC with Ni-NTA Superflow (Qiagen, Germany). A CellThru Disposable Column (Clontech, USA) was packed with the Ni-NTA slurry. The column was washed with ten volumes of buffer A containing 10 mM imidazole and 0.75 mg/ml CYMAL-5, 30 volumes of buffer A with 30 mM imidazole and 0.75 mg/ml CYMAL-5, seven volumes of buffer A with 100 mM imidazole and 0.75 mg/ ml CYMAL-5. Protein was eluted with three volumes of buffer A containing 250 mM imidazole and 0.75 mg/ml CYMAL-5 and subsequently with three volumes of buffer A with 500 mM imidazole and 0.75 mg/ml CYMAL-5. Eluted AQP1-GFP-8His protein was collected in fractions of 200 ml. All buffers contained 1 mM PMSF, 1 mg/ml Pepstatin, 1 mg/ml Chymostatin and 1 mg/ml Leupeptin.Figure 1. Structural map of the Aquaporin-1 expression plasmid pPAP8230. Abbreviations used: CYC-GALP,.
