Article open access publication

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

Scientific Reports, Springer Nature, ISSN 2045-2322

Volume 7, 1, 2017

DOI:10.1038/s41598-017-17095-6, Dimensions: pub.1093066721, PMC: PMC5715081, PMID: 29203835,

Affiliations

Organisations

  1. (1) University of Copenhagen, grid.5254.6, KU
  2. (2) Aquaporin A/S, Nymøllevej 78, 2800, Lyngby, Denmark
  3. (3) Technical University of Denmark, grid.5170.3, DTU
  4. (4) University of Maribor, grid.8647.d
  5. (5) Lund University, grid.4514.4

Countries

Denmark

Slovenia

Sweden

Continents

Europe

Description

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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NORA University Profiles

University of Copenhagen

Technical University of Denmark

Danish Open Access Indicator

2017: Realized

Research area: Science & Technology

Danish Bibliometrics Indicator

2017: Level 1

Research area: Science & Technology

Dimensions Citation Indicators

Times Cited: 14

Field Citation Ratio (FCR): 2.87

Relative Citation ratio (RCR): 1.16

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