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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 and temperature dependent accumulation of hAQP1-GFP in intact yeast cells.Briefly, yeast was inoculated in 2.5 liter shake flasks at room temperature to OD450 = 0.08 in 1 liter galactose-free expression medium (3% glycerol, 0.5% glucose minimal medium supplemented with all amino acids except leucine and isoleucine). At OD450 = 1.0 (time zero) half of the culture was transferred to 15°C and the other half to 30°C. Aquaporin expression was induced with 2% galactose 15 minutes later to assure that temperature equilibrium was obtained. Fluorescence was measured in intact yeast cells and normalized to cell number and the maximal fluorescence observed in the experiment. ○, induction of hAQP1-GFP production during growth at 15°C; •, induction of hAQP1-GFP production during growth at 30°C. Data is from a representative experiment.
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pone-0056431-g002: Time and temperature dependent accumulation of hAQP1-GFP in intact yeast cells.Briefly, yeast was inoculated in 2.5 liter shake flasks at room temperature to OD450 = 0.08 in 1 liter galactose-free expression medium (3% glycerol, 0.5% glucose minimal medium supplemented with all amino acids except leucine and isoleucine). At OD450 = 1.0 (time zero) half of the culture was transferred to 15°C and the other half to 30°C. Aquaporin expression was induced with 2% galactose 15 minutes later to assure that temperature equilibrium was obtained. Fluorescence was measured in intact yeast cells and normalized to cell number and the maximal fluorescence observed in the experiment. ○, induction of hAQP1-GFP production during growth at 15°C; •, induction of hAQP1-GFP production during growth at 30°C. Data is from a representative experiment.

Mentions: The expression studies were performed in presence of all amino acids except leucine and isoleucine as we previously described these conditions to be beneficial for recombinant membrane protein accumulation [34]. Whole cell hAQP1-GFP fluorescence was used to determine the kinetics of accumulation of functional hAQP1with respect to induction temperature. Figure 2 shows that accumulation of hAQP1-GFP increased over time and reached a plateau after 60 hours of induction at 15°C, while accumulation at 30°C peaked shortly (≈12 hours) after induction and subsequently decreased. Expression at 15°C was therefore favorable for production of hAQP1-GFP.


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 and temperature dependent accumulation of hAQP1-GFP in intact yeast cells.Briefly, yeast was inoculated in 2.5 liter shake flasks at room temperature to OD450 = 0.08 in 1 liter galactose-free expression medium (3% glycerol, 0.5% glucose minimal medium supplemented with all amino acids except leucine and isoleucine). At OD450 = 1.0 (time zero) half of the culture was transferred to 15°C and the other half to 30°C. Aquaporin expression was induced with 2% galactose 15 minutes later to assure that temperature equilibrium was obtained. Fluorescence was measured in intact yeast cells and normalized to cell number and the maximal fluorescence observed in the experiment. ○, induction of hAQP1-GFP production during growth at 15°C; •, induction of hAQP1-GFP production during growth at 30°C. Data is from a representative experiment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056431-g002: Time and temperature dependent accumulation of hAQP1-GFP in intact yeast cells.Briefly, yeast was inoculated in 2.5 liter shake flasks at room temperature to OD450 = 0.08 in 1 liter galactose-free expression medium (3% glycerol, 0.5% glucose minimal medium supplemented with all amino acids except leucine and isoleucine). At OD450 = 1.0 (time zero) half of the culture was transferred to 15°C and the other half to 30°C. Aquaporin expression was induced with 2% galactose 15 minutes later to assure that temperature equilibrium was obtained. Fluorescence was measured in intact yeast cells and normalized to cell number and the maximal fluorescence observed in the experiment. ○, induction of hAQP1-GFP production during growth at 15°C; •, induction of hAQP1-GFP production during growth at 30°C. Data is from a representative experiment.
Mentions: The expression studies were performed in presence of all amino acids except leucine and isoleucine as we previously described these conditions to be beneficial for recombinant membrane protein accumulation [34]. Whole cell hAQP1-GFP fluorescence was used to determine the kinetics of accumulation of functional hAQP1with respect to induction temperature. Figure 2 shows that accumulation of hAQP1-GFP increased over time and reached a plateau after 60 hours of induction at 15°C, while accumulation at 30°C peaked shortly (≈12 hours) after induction and subsequently decreased. Expression at 15°C was therefore favorable for production of hAQP1-GFP.

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