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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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Endo glycosidase H treatment of yeast crude membranes.÷, crude membranes from yeast producing hAQP1-GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His.
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pone-0056431-g005: Endo glycosidase H treatment of yeast crude membranes.÷, crude membranes from yeast producing hAQP1-GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His.

Mentions: In erythrocytes hAQP1is found in two forms; a non-glycosylated version and an extensively N-glycosylated form [13]. To analyze whether recombinant hAQP1-GFP-8His is N-glycosylated we separated crude membranes treated or not with Endo-glycosidase H by SDS-PAGE an analyzed the outcome by in-gel fluorescence. The data in Figure 5 show that EndoH treatment did not affect the electrophoretic mobility of hAQP1-GFP-His8 showing that the fusion protein was not N-glycosylated.


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)

Endo glycosidase H treatment of yeast crude membranes.÷, crude membranes from yeast producing hAQP1-GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056431-g005: Endo glycosidase H treatment of yeast crude membranes.÷, crude membranes from yeast producing hAQP1-GFP-8His; +, Endo-H treatment of crude membranes from yeast producing hAQP1-GFP-8His.
Mentions: In erythrocytes hAQP1is found in two forms; a non-glycosylated version and an extensively N-glycosylated form [13]. To analyze whether recombinant hAQP1-GFP-8His is N-glycosylated we separated crude membranes treated or not with Endo-glycosidase H by SDS-PAGE an analyzed the outcome by in-gel fluorescence. The data in Figure 5 show that EndoH treatment did not affect the electrophoretic mobility of hAQP1-GFP-His8 showing that the fusion protein was not N-glycosylated.

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.

Show MeSH
Related in: MedlinePlus