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

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.

Krabbe, Simon LyngaaUniversity of Copenhagen (författare)
Nurup, Casper NormannUniversity of Copenhagen (författare)
Missel, Julie WinkelUniversity of Copenhagen (författare)
Spulber, MarianaAquaporin A/S (författare)
Bomholt, JulieAquaporin A/S (författare)
Molbaek, KarenUniversity of Copenhagen (författare)
Helix-Nielsen, ClausUniversity of Maribor,Technical University of Denmark,Aquaporin A/S (författare)
Gotfryd, KamilUniversity of Copenhagen (författare)
Gourdon, PontusLund University,Lunds universitet,Membranproteinstrukturbiologi,Forskargrupper vid Lunds universitet,Membrane Protein Structural Biology,Lund University Research Groups,University of Copenhagen(Swepub:lu)med-pgd (författare)
Pedersen, Per AmstrupUniversity of Copenhagen (författare)
University of CopenhagenAquaporin A/S (creator_code:org_t)

Av författaren/redakt...: Bjørkskov, Frede ...; Krabbe, Simon Ly ...; Nurup, Casper No ...; Missel, Julie Wi ...; Spulber, Mariana; Bomholt, Julie; visa fler...; Molbaek, Karen; Helix-Nielsen, C ...; Gotfryd, Kamil; Gourdon, Pontus; Pedersen, Per Am ...; visa färre...

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