Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiae.
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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Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, Gif sur Yvette, France.
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Tacnet
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D000911:Antibodies, Monoclonal; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D011510:Proteolipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016324:Sequence Tagged Sites; D013816:Thermodynamics
C496020:AQP1 protein, human; D000911:Antibodies, Monoclonal; D020346:Aquaporins; D001789:Blood Group Antigens; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007473:Ion Channels; D008081:Liposomes; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C020485:proteoliposomes; D051398:Aquaporin 1