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.
MeSH terms
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Antibodies, Monoclonal
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Aquaporin 1
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Aquaporins*
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Blood Group Antigens
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Chromatography, Affinity
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Cloning, Molecular / methods
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Hemagglutinin Glycoproteins, Influenza Virus / biosynthesis
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Humans
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Ion Channels / biosynthesis*
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Ion Channels / isolation & purification*
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Ion Channels / metabolism
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Kinetics
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Liposomes
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Proteolipids / metabolism
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae
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Sequence Tagged Sites
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Thermodynamics
Substances
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AQP1 protein, human
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Antibodies, Monoclonal
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Aquaporins
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Blood Group Antigens
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Hemagglutinin Glycoproteins, Influenza Virus
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Ion Channels
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Liposomes
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Proteolipids
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Recombinant Fusion Proteins
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Recombinant Proteins
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proteoliposomes
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Aquaporin 1