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

Per Amstrup Pedersen, Pontus Emanuel Gourdon, Kamil Gotfryd, Karen Molbæk Hansen, Claus Hélix-Nielsen, Julie Bomholt, Mariana Spulber, Julie Winkel Missel, Frederik Bühring Bjørkskov, Simon Lyngaa Krabbe, Casper Normann Nurup

Research output: Contribution to journalJournal articleResearchpeer-review

273 Downloads (Pure)

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 languageEnglish
Article number16899
JournalScientific Reports
Volume7
Number of pages21
ISSN2045-2322
DOIs
Publication statusPublished - 2017

Bibliographical note

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Fingerprint Dive into the research topics of 'Purification and functional characterization of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization'. Together they form a unique fingerprint.

Cite this