PURIFICATION AND FUNCTIONAL RECONSTITUTION OF THE HUMAN CHIP28 WATER CHANNEL EXPRESSED IN SACCHAROMYCES-CEREVISIAE

The yeast Saccharomyces cerevisiae was used for heterologous expressio n of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine- aminoacid 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 extr acts by immunochromatography on protein A/12CA5-coupled beads, after K I extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteol iposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capa city to produce functional aquaporins at levels sufficient for biochem ical and biophysical analyses. (C) 1997 Academic Press.