Tandem affinity purification of the Candida albicans septin protein complex
Tandem affinity purification of the Candida albicans septin protein complex
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作者：
Kaneko, A;Umeyama, T;Niimi, M
构建了一种新的载体，使单个基因的整合标记和亲和纯化的相互作用的分子内的蛋白质复合物从白色念珠菌使用串联6x组氨酸和FLAG表位标签。通过纯化C.白色念珠菌septin复合物（一种细胞骨架蛋白的自缔合复合物），来自酵母和菌丝细胞。一步亲和纯化不足以纯化的蛋白质复合物，而串联亲和纯化（TAP）得到了广泛纯化的蛋白质复合物具有非常低的背景。电泳和质谱分析表明，亲和纯化的C。主要由CaCdc 3 p、CaCdc 10 p、CaCdc 11 p、CaCdc 12 p和CaSep 7 p组成的白念珠菌septin复合物不受细胞形态的影响。纯化的septin复合物似乎具有2 CaCdc 3 p，1-2 CaCdc 10 p，1 CaCdc 11 p，2 CaCdc 12 p和小于或等于1 CaSep 7 p的化学计量。TAP在分离纯化和分析C.白念珠菌Septin复合物表明该技术将在该病原真菌的蛋白质组学研究中有更广泛的应用。版权所有（C）2004约翰威利父子有限公司。
A novel vector was constructed to enable the integrative marking of individual genes and the affinity purification of interacting molecules within protein complexes from Candida albicans using a tandem 6xhistidine and FLAG epitope tag. The system was verified by purifying the C. albicans septin complex (a self-associating complex of cytoskeletal proteins) from both yeast and hyphal cells. One-step affinity purification was insufficient for purification of the protein complex, whereas tandem affinity purification (TAP) gave an extensively purified protein complex with a very low background. Electrophoretic and mass spectrometry analysis showed that the affinity-purified C. albicans septin complex, which comprises predominantly CaCdc3p, CaCdc10p, CaCdc11p, CaCdc12p and CaSep7p, was not affected by cell morphology. The purified septin complex appeared to have a stoichiometry of 2 CaCdc3p, 1-2 CaCdc10p, 1 CaCdc11p, 2 CaCdc12p and less than or equal to1 CaSep7p. The successful application of TAP to the purification and analysis of the C. albicans septin complex indicates that this technology will have much wider application to proteomic studies of this pathogenic fungus. Copyright (C) 2004 John Wiley Sons, Ltd.