Luted at an imidazol concentration of 250 mM. Ingel fluorescence followed by Coomassie staining showed that the protein eluted as a monomer, dimer, trimer and tetramer as seen for purification of the native protein from erythrocytes [48]. The Coomassie stain shows that solubilization in CYMAL-5 followed by Ni-affinity chromatography resulted in a very pure preparation of recombinant hAQP1-GFP-8His fusion protein. Comparing theFigure 9. Affinity purification of hAQP1-GFP-8His. Crude membranes were solubilized in CYMAL-5 and purified by Ni-affinity chromatography as described in Materials and Methods. A, GFP fluorescence (red) was used to quantify the amount of hAQP1 in each fraction. The Imidazol profile used to wash and elute protein from the Ni-column is shown in blue. AU, arbitrary fluorescence units. B, (1) in-gel fluorescence after SDS-PAGE separation of the protein content 23727046 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flowthrough; fractions 1- 3, wash with 10 mM Imidazole; fractions 4?1 wash with 30 mM Imidazole; fractions 12?0, wash with 100 mM Imidazole; fractions 21?5, wash with 250 mM Imidazole; fractions 26?0, wash with 500 mM Imidazole. doi:10.1371/journal.pone.0056431.gHigh Level Human Aquaporin Production in Yeastin-gel fluorescence with the Coomassie stain (Figure 7) also indicates that the purified hAQP1-GFP-8His fusion proteins are correctly folded since only bands detected by in-gel fluorescence were visible in the Coomassie stain. The slower migrating and non-fluorescent hAQP1-GFP-8His fusion proteins present in the western blot in Figure 3 were absent in the purified preparation. In contrast to Aquaporin-1 from erythrocytes we showed that the recombinantly produced protein in yeast was not N-glycosylated. In conclusion we have developed an expression system that substantially increases the membrane density of recombinant hAQP1.This expression system enables low cost production of large amounts of functional protein for structural and biophysical studies and may become an important tool for identification of hAQP1 modulators.AcknowledgmentsThe authors thank David S ensen for excellent technical assistance, Dr. David Drew for generous gift of the GFP expression plasmid, pET20bGFP-8His and Dr. Jakob Winther for the anti-GFP ntibody.Author ContributionsConceived and Peptide M web designed the experiments: JB PSP PAP. Performed the experiments: JB PSP PAP. Analyzed the data: JB CHN PSP PAP. Contributed reagents/materials/analysis tools: JB PSP CHN PAP. Wrote the paper: JB CHN PAP.
Illicit stimulants such as amphetamine, methamphetamine, cocaine, and ecstasy (3,4-methylenedioxymethMedChemExpress Thiazole Orange amphetamine or MDMA) temporarily increase alertness, mood, and euphoria. These effects arise from their acute mechanism of action on the monoamine neurotransmitters dopamine, noradrenaline, and serotonin. There are important differences in the degree to which the different stimulants affect these three neurotransmitters. For example, amphetamine, methamphetamine, and cocaine administration all result in excess accumulation of mainly dopamine [1,2,3] whereas ecstasy administration results in accumulation of mainly serotonin and noradrenaline [4]. Animal and in vitro studies show that amphetamine and methamphetamine disrupt synaptic vesicles, inhibit monoamine oxidase [5,6], and block and/ or reverse vesicular monoamine transporters [7,8]. Furthermore, both amphetamines and cocaine affect dopamin.Luted at an imidazol concentration of 250 mM. Ingel fluorescence followed by Coomassie staining showed that the protein eluted as a monomer, dimer, trimer and tetramer as seen for purification of the native protein from erythrocytes [48]. The Coomassie stain shows that solubilization in CYMAL-5 followed by Ni-affinity chromatography resulted in a very pure preparation of recombinant hAQP1-GFP-8His fusion protein. Comparing theFigure 9. Affinity purification of hAQP1-GFP-8His. Crude membranes were solubilized in CYMAL-5 and purified by Ni-affinity chromatography as described in Materials and Methods. A, GFP fluorescence (red) was used to quantify the amount of hAQP1 in each fraction. The Imidazol profile used to wash and elute protein from the Ni-column is shown in blue. AU, arbitrary fluorescence units. B, (1) in-gel fluorescence after SDS-PAGE separation of the protein content 23727046 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flowthrough; fractions 1- 3, wash with 10 mM Imidazole; fractions 4?1 wash with 30 mM Imidazole; fractions 12?0, wash with 100 mM Imidazole; fractions 21?5, wash with 250 mM Imidazole; fractions 26?0, wash with 500 mM Imidazole. doi:10.1371/journal.pone.0056431.gHigh Level Human Aquaporin Production in Yeastin-gel fluorescence with the Coomassie stain (Figure 7) also indicates that the purified hAQP1-GFP-8His fusion proteins are correctly folded since only bands detected by in-gel fluorescence were visible in the Coomassie stain. The slower migrating and non-fluorescent hAQP1-GFP-8His fusion proteins present in the western blot in Figure 3 were absent in the purified preparation. In contrast to Aquaporin-1 from erythrocytes we showed that the recombinantly produced protein in yeast was not N-glycosylated. In conclusion we have developed an expression system that substantially increases the membrane density of recombinant hAQP1.This expression system enables low cost production of large amounts of functional protein for structural and biophysical studies and may become an important tool for identification of hAQP1 modulators.AcknowledgmentsThe authors thank David S ensen for excellent technical assistance, Dr. David Drew for generous gift of the GFP expression plasmid, pET20bGFP-8His and Dr. Jakob Winther for the anti-GFP ntibody.Author ContributionsConceived and designed the experiments: JB PSP PAP. Performed the experiments: JB PSP PAP. Analyzed the data: JB CHN PSP PAP. Contributed reagents/materials/analysis tools: JB PSP CHN PAP. Wrote the paper: JB CHN PAP.
Illicit stimulants such as amphetamine, methamphetamine, cocaine, and ecstasy (3,4-methylenedioxymethamphetamine or MDMA) temporarily increase alertness, mood, and euphoria. These effects arise from their acute mechanism of action on the monoamine neurotransmitters dopamine, noradrenaline, and serotonin. There are important differences in the degree to which the different stimulants affect these three neurotransmitters. For example, amphetamine, methamphetamine, and cocaine administration all result in excess accumulation of mainly dopamine [1,2,3] whereas ecstasy administration results in accumulation of mainly serotonin and noradrenaline [4]. Animal and in vitro studies show that amphetamine and methamphetamine disrupt synaptic vesicles, inhibit monoamine oxidase [5,6], and block and/ or reverse vesicular monoamine transporters [7,8]. Furthermore, both amphetamines and cocaine affect dopamin.
