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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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In-gel fluorescence and western bloting of yeast membranes.A, in-gel fluorescence of SDS-PAGE separated crude membranes isolated from yeast expressing the AQP1-GFP fusion for the times indicated at 15°C or 30°C. B, western blotting of the gels shown in ‘A’ using an anti-GFP-antibody. Folding efficiency at each time point and temperature was estimated as the ratio between the intensity of correctly folded hAQP-1-GFP fusion and the sum of intensities for the correctly folded and mal-folded GFP-fusions. MFP, mal-folded protein; CFP, correctly folded protein. The data are from a representative experiment.
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pone-0056431-g003: In-gel fluorescence and western bloting of yeast membranes.A, in-gel fluorescence of SDS-PAGE separated crude membranes isolated from yeast expressing the AQP1-GFP fusion for the times indicated at 15°C or 30°C. B, western blotting of the gels shown in ‘A’ using an anti-GFP-antibody. Folding efficiency at each time point and temperature was estimated as the ratio between the intensity of correctly folded hAQP-1-GFP fusion and the sum of intensities for the correctly folded and mal-folded GFP-fusions. MFP, mal-folded protein; CFP, correctly folded protein. The data are from a representative experiment.

Mentions: To identify the molecular mechanism behind temperature sensitive accumulation of hAQP1-GFP we isolated membranes from yeast cells expressing the GFP fusion at either 15°C or 30°C and analyzed the purified membranes by in-gel fluorescence and western blotting. Only correctly folded GFP is visualized by in-gel fluorescence while correctly folded as well as mal-folded GFP are recognized by the anti-GFP-antibody in western blots. In the SDS-PAGE gel the Aquaporin-1 part of the fusion is denatured while the compact structure of correctly folded GFP is resistant to the applied SDS concentration [36]. The electrophoretic mobility of Aquaporin-1 fused to correctly folded GFP is therefore increased compared to that of Aquaporin-1 fused to mal-folded GFP. The in-gel fluorescence data in Figure 3A show that only a single membrane protein of approximately 40 kDa is visible after expression at 15°C and 30°C. The electrophoretic mobility of this band is in accordance with the expected molecular weight of the fluorescent band since hAQP1 has a molecular weight of 28.5 kDa and correctly folded GFP increases the molecular weight with 10–15 kDa [36] while the His-tag contributes with 1.1 kDa. The western blot data in Figure 3B show that the hAQP1-GFP-8His protein accumulated as a fast migrating correctly folded protein as well as a slower migrating mal-folded protein. Quantification of the data in Figure 3B show that up till 90% of hAQP-1 protein was correctly folded at 15°C while approximately 25% was correctly folded at 30°C.


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)

In-gel fluorescence and western bloting of yeast membranes.A, in-gel fluorescence of SDS-PAGE separated crude membranes isolated from yeast expressing the AQP1-GFP fusion for the times indicated at 15°C or 30°C. B, western blotting of the gels shown in ‘A’ using an anti-GFP-antibody. Folding efficiency at each time point and temperature was estimated as the ratio between the intensity of correctly folded hAQP-1-GFP fusion and the sum of intensities for the correctly folded and mal-folded GFP-fusions. MFP, mal-folded protein; CFP, correctly folded protein. The data are from a representative experiment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569440&req=5

pone-0056431-g003: In-gel fluorescence and western bloting of yeast membranes.A, in-gel fluorescence of SDS-PAGE separated crude membranes isolated from yeast expressing the AQP1-GFP fusion for the times indicated at 15°C or 30°C. B, western blotting of the gels shown in ‘A’ using an anti-GFP-antibody. Folding efficiency at each time point and temperature was estimated as the ratio between the intensity of correctly folded hAQP-1-GFP fusion and the sum of intensities for the correctly folded and mal-folded GFP-fusions. MFP, mal-folded protein; CFP, correctly folded protein. The data are from a representative experiment.
Mentions: To identify the molecular mechanism behind temperature sensitive accumulation of hAQP1-GFP we isolated membranes from yeast cells expressing the GFP fusion at either 15°C or 30°C and analyzed the purified membranes by in-gel fluorescence and western blotting. Only correctly folded GFP is visualized by in-gel fluorescence while correctly folded as well as mal-folded GFP are recognized by the anti-GFP-antibody in western blots. In the SDS-PAGE gel the Aquaporin-1 part of the fusion is denatured while the compact structure of correctly folded GFP is resistant to the applied SDS concentration [36]. The electrophoretic mobility of Aquaporin-1 fused to correctly folded GFP is therefore increased compared to that of Aquaporin-1 fused to mal-folded GFP. The in-gel fluorescence data in Figure 3A show that only a single membrane protein of approximately 40 kDa is visible after expression at 15°C and 30°C. The electrophoretic mobility of this band is in accordance with the expected molecular weight of the fluorescent band since hAQP1 has a molecular weight of 28.5 kDa and correctly folded GFP increases the molecular weight with 10–15 kDa [36] while the His-tag contributes with 1.1 kDa. The western blot data in Figure 3B show that the hAQP1-GFP-8His protein accumulated as a fast migrating correctly folded protein as well as a slower migrating mal-folded protein. Quantification of the data in Figure 3B show that up till 90% of hAQP-1 protein was correctly folded at 15°C while approximately 25% was correctly folded at 30°C.

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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