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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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Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1.(A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence. All images were generated at 100 times magnification.
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pone-0056431-g006: Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1.(A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence. All images were generated at 100 times magnification.

Mentions: Bioimaging of live yeast cells expressing hAQP1-GFP-8His was used to determine the sub cellular localization of the recombinant protein in yeast. Cells were additionally stained with DAPI to localize the nucleus and with FM4-64 that under the conditions used in the present protocol colors the vacuole as well as the plasma membrane. It can be seen from the micrographs in Figure 6 that a major part of hAQP1-GFP-8His was located non-uniformly in the plasma membrane; possibly indicating localization in lipid rafts. A part of the GFP fusion is also observed to localize in internal membranes, probably Endoplasmic Reticulum.


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)

Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1.(A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence. All images were generated at 100 times magnification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056431-g006: Live cell bioimaging of S. cerevisiae expressing GFP tagged hAQP1.(A), phase contrast; (B), GFP fluorescence; (C), DAPI fluorescence; (D), FM4-64 fluorescence. All images were generated at 100 times magnification.
Mentions: Bioimaging of live yeast cells expressing hAQP1-GFP-8His was used to determine the sub cellular localization of the recombinant protein in yeast. Cells were additionally stained with DAPI to localize the nucleus and with FM4-64 that under the conditions used in the present protocol colors the vacuole as well as the plasma membrane. It can be seen from the micrographs in Figure 6 that a major part of hAQP1-GFP-8His was located non-uniformly in the plasma membrane; possibly indicating localization in lipid rafts. A part of the GFP fusion is also observed to localize in internal membranes, probably Endoplasmic Reticulum.

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