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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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Fluorescent-detection size-exclusion chromatography of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM.Crude membranes solubilized in CYMAL-5 or DDM at a detergent: protein ratio of 3 were separated by HPLC on a SuperoseTM 12 10/300 GL column. Fractions of 300 µl were collected from the column and analyzed for GFP fluorescence as described in Materials and Methods.
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pone-0056431-g008: Fluorescent-detection size-exclusion chromatography of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM.Crude membranes solubilized in CYMAL-5 or DDM at a detergent: protein ratio of 3 were separated by HPLC on a SuperoseTM 12 10/300 GL column. Fractions of 300 µl were collected from the column and analyzed for GFP fluorescence as described in Materials and Methods.

Mentions: A suitable detergent efficiently solubilizes the membrane protein of interest, gives a monodisperse protein preparation and maintains protein stability over a long period of time. To analyze for these quality criteria we examined the fusion protein by fluorescent size exclusion chromatography (FSEC) [43] of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM to analyze for monodispersity. Data in Figure 8 show that the two detergents gave rise to similar chromatograms; a prominent symmetrical peak eluting in a total volume of less than 2 ml indicating a monodisperse protein preparation [35]. The elution volume corresponds to a molecular weight of approximately 200 kDa and indicates that solubilized, recombinant hAQP1-GFP-8His mainly exists as a tetramer in both detergents. Presence of a minor symmetrical peak shows that hAQP1-GFP-8His oligomers with a stoichiometry larger than four also exist. There is no sign of protein degradation as no free-GFP peak is visible.


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)

Fluorescent-detection size-exclusion chromatography of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM.Crude membranes solubilized in CYMAL-5 or DDM at a detergent: protein ratio of 3 were separated by HPLC on a SuperoseTM 12 10/300 GL column. Fractions of 300 µl were collected from the column and analyzed for GFP fluorescence as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056431-g008: Fluorescent-detection size-exclusion chromatography of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM.Crude membranes solubilized in CYMAL-5 or DDM at a detergent: protein ratio of 3 were separated by HPLC on a SuperoseTM 12 10/300 GL column. Fractions of 300 µl were collected from the column and analyzed for GFP fluorescence as described in Materials and Methods.
Mentions: A suitable detergent efficiently solubilizes the membrane protein of interest, gives a monodisperse protein preparation and maintains protein stability over a long period of time. To analyze for these quality criteria we examined the fusion protein by fluorescent size exclusion chromatography (FSEC) [43] of hAQP1-GFP-8His solubilized in CYMAL-5 or DDM to analyze for monodispersity. Data in Figure 8 show that the two detergents gave rise to similar chromatograms; a prominent symmetrical peak eluting in a total volume of less than 2 ml indicating a monodisperse protein preparation [35]. The elution volume corresponds to a molecular weight of approximately 200 kDa and indicates that solubilized, recombinant hAQP1-GFP-8His mainly exists as a tetramer in both detergents. Presence of a minor symmetrical peak shows that hAQP1-GFP-8His oligomers with a stoichiometry larger than four also exist. There is no sign of protein degradation as no free-GFP peak is visible.

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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