PURIFICATION OF A RAS-RESPONSIVE ADENYLYL CYCLASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE BY USE OF AN EPITOPE ADDITION METHOD
PURIFICATION OF A RAS-RESPONSIVE ADENYLYL CYCLASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE BY USE OF AN EPITOPE ADDITION METHOD
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DOI：
10.1128/mcb.8.5.2159
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发表时间：
1988-05-01
影响因子：
5.3
通讯作者：
WIGLER, M
中科院分区：
文献类型：
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作者：
FIELD, J;NIKAWA, J;WIGLER, M
我们建立了一种基于与小肽表位融合的免疫亲和纯化酿酒酵母腺苷酸环化酶的方法。利用寡核苷酸技术编码肽表位，构建了表达融合蛋白的质粒。cereviasiae乙醇脱氢酶启动子ADH 1。使用先前针对肽产生的单克隆抗体来纯化由200千道尔顿腺苷酸环化酶融合蛋白和未鉴定的70千道尔顿蛋白组成的肽。纯化的蛋白质可被RAS蛋白激活。激活绝对需要鸟嘌呤核苷三磷酸。
We developed a method for immunoaffinity purfication of Scaccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cereviasiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.