Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiae

Protein Expression and Purification
V LaizéF Tacnet

Abstract

The yeast Saccharomyces cerevisiae was used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/ 12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.

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Citations

Feb 3, 1999·Annual Review of Genetics·J W Jarvik, C A Telmer
Apr 15, 2011·American Journal of Physiology. Renal Physiology·Alexander R KolbJeffrey L Brodsky
Dec 9, 2014·Acta Crystallographica. Section F, Structural Biology Communications·David Ruiz CarrilloJulien Lescar
Oct 29, 2005·Journal of Molecular Microbiology and Biotechnology·An TanghePatrick Van Dijck
Feb 8, 2000·American Journal of Physiology. Renal Physiology·A S Verkman, A K Mitra
Mar 27, 1999·Biochemical and Biophysical Research Communications·V LaizéF Tacnet
Sep 18, 2007·Protein Expression and Purification·Maria NyblomKristina Hedfalk

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