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Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

Bomholt J, Hélix-Nielsen C, Scharff-Poulsen P, Pedersen PA - PLoS ONE (2013)

Bottom Line: Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium.A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation.Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: Aquaporin A/S, Copenhagen, Denmark.

ABSTRACT
In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator 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 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 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. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

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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 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flow-through; fractions 1- 3, wash with 10 mM Imidazole; fractions 4–11 wash with 30 mM Imidazole; fractions 12–20, wash with 100 mM Imidazole; fractions 21–25, wash with 250 mM Imidazole; fractions 26–30, wash with 500 mM Imidazole.
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pone-0056431-g009: 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 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flow-through; fractions 1- 3, wash with 10 mM Imidazole; fractions 4–11 wash with 30 mM Imidazole; fractions 12–20, wash with 100 mM Imidazole; fractions 21–25, wash with 250 mM Imidazole; fractions 26–30, wash with 500 mM Imidazole.

Mentions: CYMAL-5 efficiently solubilized hAQP1-GFP-His8 and produced a monodisperse protein preparation mainly consisting of the native tetrameric structure. We therefore selected CYMAL-5 solubilized hAQP1-GFP-8His for purification by affinity chromatography. A chromatogram resulting from the purification procedure is shown in Figure 9A. Data from the purification protocol revealed that 86% of the CYMAL-5 solubilized hAQP1-GFP-8His protein bound to the Ni2+-resin and 62% of the solubilized and bound material was eluted with 250 mM imidazole. The peak-fraction collected after elution with 250 mM imidazole was analyzed by SDS-PAGE separation using in-gel fluorescence and Coomassie staining, Figure 9B. As expected monomeric, dimeric, trimeric as well as tetrameric hAQP1-GFP-8His proteins were visible. The Coomassie staining furthermore showed that solubilization of the hAQP1-GFP-8His protein by CYMAL-5 followed by Ni-affinity chromatography resulted in highly pure human Aquaporin-1 protein. Very importantly only protein bands visualized by in-gel fluorescence were observed in the Coomassie stain. None of the slower migrating, non-fluorescent and mal-folded hAQP1-GFP-8His proteins observed in the western blot in Figure 3 were present in the purified sample.


Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

Bomholt J, Hélix-Nielsen C, Scharff-Poulsen P, Pedersen PA - PLoS ONE (2013)

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 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flow-through; fractions 1- 3, wash with 10 mM Imidazole; fractions 4–11 wash with 30 mM Imidazole; fractions 12–20, wash with 100 mM Imidazole; fractions 21–25, wash with 250 mM Imidazole; fractions 26–30, wash with 500 mM Imidazole.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569440&req=5

pone-0056431-g009: 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 of fraction 22; (2), Coomassie staining of the SDS-PAGE gel used for in-gel fluorescence in panel (1). Fraction 0, flow-through; fractions 1- 3, wash with 10 mM Imidazole; fractions 4–11 wash with 30 mM Imidazole; fractions 12–20, wash with 100 mM Imidazole; fractions 21–25, wash with 250 mM Imidazole; fractions 26–30, wash with 500 mM Imidazole.
Mentions: CYMAL-5 efficiently solubilized hAQP1-GFP-His8 and produced a monodisperse protein preparation mainly consisting of the native tetrameric structure. We therefore selected CYMAL-5 solubilized hAQP1-GFP-8His for purification by affinity chromatography. A chromatogram resulting from the purification procedure is shown in Figure 9A. Data from the purification protocol revealed that 86% of the CYMAL-5 solubilized hAQP1-GFP-8His protein bound to the Ni2+-resin and 62% of the solubilized and bound material was eluted with 250 mM imidazole. The peak-fraction collected after elution with 250 mM imidazole was analyzed by SDS-PAGE separation using in-gel fluorescence and Coomassie staining, Figure 9B. As expected monomeric, dimeric, trimeric as well as tetrameric hAQP1-GFP-8His proteins were visible. The Coomassie staining furthermore showed that solubilization of the hAQP1-GFP-8His protein by CYMAL-5 followed by Ni-affinity chromatography resulted in highly pure human Aquaporin-1 protein. Very importantly only protein bands visualized by in-gel fluorescence were observed in the Coomassie stain. None of the slower migrating, non-fluorescent and mal-folded hAQP1-GFP-8His proteins observed in the western blot in Figure 3 were present in the purified sample.

Bottom Line: Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium.A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation.Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: Aquaporin A/S, Copenhagen, Denmark.

ABSTRACT
In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator 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 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 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. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

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Related in: MedlinePlus