Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization

Frederik Bühring Bjørkskov; Simon Krabbe; Casper Normann Nurup; Julie Winkel Missel; Mariana Spulber; Julie Bomholt; Karen Molbaek; Claus Helix-Nielsen; Kamil Gotfryd; Pontus Emanuel Gourdon; Per Amstrup Pedersen

doi:10.1038/s41598-017-17095-6

Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization

Frederik Bühring Bjørkskov, Simon Krabbe, Casper Normann Nurup, Julie Winkel Missel, Mariana Spulber, Julie Bomholt, Karen Molbaek, Claus Helix-Nielsen, Kamil Gotfryd, Pontus Emanuel Gourdon, Per Amstrup Pedersen^*

^*Corresponding author for this work

18 Citations (Scopus)

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Abstract

The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.

Original language	English
Article number	16899
Journal	Scientific Reports
Volume	7
Number of pages	21
ISSN	2045-2322
DOIs	https://doi.org/10.1038/s41598-017-17095-6
Publication status	Published - 4 Dec 2017

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Bjørkskov, F. B., Krabbe, S., Nurup, C. N., Missel, J. W., Spulber, M., Bomholt, J., Molbaek, K., Helix-Nielsen, C., Gotfryd, K., Gourdon, P. E., & Pedersen, P. A. (2017). Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization. Scientific Reports, 7, [16899]. https://doi.org/10.1038/s41598-017-17095-6

Bjørkskov, FB, Krabbe, S, Nurup, CN, Missel, JW, Spulber, M, Bomholt, J, Molbaek, K, Helix-Nielsen, C, Gotfryd, K, Gourdon, PE & Pedersen, PA 2017, 'Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization', Scientific Reports, vol. 7, 16899. https://doi.org/10.1038/s41598-017-17095-6

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title = "Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization",

abstract = "The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.",

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AU - Missel, Julie Winkel

AU - Spulber, Mariana

AU - Bomholt, Julie

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AU - Helix-Nielsen, Claus

AU - Gotfryd, Kamil

AU - Gourdon, Pontus Emanuel

AU - Pedersen, Per Amstrup

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N2 - The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.

AB - The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization.

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Purification and functional comparison of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization

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