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.
Highlights
The great majority of membrane protein structures originates from prokaryotes and only 59 are of human origin[2]
The road to more high resolution structures of human membrane proteins is, paved with severe obstacles related to difficulties in achieving high-level high quality expression, and establishing efficient purification procedures that retain the biological activity of the protein, thereby securing samples suitable for structural biology methods such as X-ray crystallography and cryo-EM
Thirteen different AQPs have been identified in humans[12]. These are traditionally subdivided into three major groups; the orthodox AQPs; AQP0, AQP1, AQP2, AQP4, AQP5, AQP6 and AQP8; the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 and the super-AQPs, AQP11 and AQP1213
Summary
The great majority of membrane protein structures originates from prokaryotes and only 59 are of human origin[2]. The road to more high resolution structures of human membrane proteins is, paved with severe obstacles related to difficulties in achieving high-level high quality expression, and establishing efficient purification procedures that retain the biological activity of the protein, thereby securing samples suitable for structural biology methods such as X-ray crystallography and cryo-EM. Dealing with these issues is far from being trivial and usually approached by a laborious trial and error strategy. A primary structure alignment of the AQPs studied in the present paper is shown in Supplementary Figure S2.The molecular weight of each monomer ranges from 27 kDa to 37 kDa depending on the AQP (http://www.uniprot.org/).We show high yields of most targets as wells as a characterization of their water and glycerol permeabilities
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