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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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Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes.A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15°C at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose.
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pone-0056431-g004: Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes.A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15°C at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose.

Mentions: Quantification of the in-gel fluorescence data in Figure 3A showed that correctly folded hAQP1-GFP accumulated in yeast membranes at 15°C even up till 124 hours after induction (data not shown). In order to determine if the membrane density of hAQP1-GFP protein kept increasing we quantified the time dependent accumulation of hAQP1-GFP produced at 15°C in crude membranes. It can be seen from Figure 4 that the density continued to increase for at least up till two weeks after induction, and that the density reached almost 1,500 pmol hAQP1-GFP per mg crude membranes. This corresponds to 8.5% of the total membrane protein content. The intense green color emitted from the crude membrane preparation shown in Figure 4 visualizes the high membrane density of the hAQP1-GFP fusion protein.


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)

Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes.A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15°C at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056431-g004: Time dependent accumulation of hAQP1-GFP fluorescence in crude membranes.A, hAQP1-GFP fluorescence in crude membranes isolated from yeast grown at 15°C at different time points after induction with galactose at time zero. Fluorescence intensity was converted to pmol GFP/mg crude membrane protein using a standard curve generated from purified yeGFP. B, crude membranes at a concentration of 6 mg/ml isolated 336 hours after induction with galactose.
Mentions: Quantification of the in-gel fluorescence data in Figure 3A showed that correctly folded hAQP1-GFP accumulated in yeast membranes at 15°C even up till 124 hours after induction (data not shown). In order to determine if the membrane density of hAQP1-GFP protein kept increasing we quantified the time dependent accumulation of hAQP1-GFP produced at 15°C in crude membranes. It can be seen from Figure 4 that the density continued to increase for at least up till two weeks after induction, and that the density reached almost 1,500 pmol hAQP1-GFP per mg crude membranes. This corresponds to 8.5% of the total membrane protein content. The intense green color emitted from the crude membrane preparation shown in Figure 4 visualizes the high membrane density of the hAQP1-GFP fusion protein.

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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