Laizé V, Ripoche P, Tacnet F
Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, Gif sur Yvette, France.
Protein Expr Purif. 1997 Dec;11(3):284-8. doi: 10.1006/prep.1997.0798.
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.
酿酒酵母被用于人CHIP28水通道蛋白-1（水通道蛋白-1）的异源表达。选择了单克隆抗体12CA5识别的流感血凝素蛋白的九氨基酸表位（HA表位），用于在CHIP28的N端进行标记。在碘化钾提取和去污剂增溶后，通过在蛋白A/12CA5偶联磁珠上进行免疫色谱，从酵母提取物中纯化表位标记的CHIP28，然后通过阴离子交换色谱进行浓缩。纯化后的蛋白被重组到蛋白脂质体中，并通过停流分光光度法显示其具有水通道功能。这项研究表明，酵母有能力产生足够用于生化和生物物理分析的功能性水通道蛋白。
