Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.
Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.
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DOI：
10.1021/bi00145a019
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发表时间：
1992-08
期刊：
影响因子：
2.9
通讯作者：
N. Thompson;D. Hager;R. Burgess
中科院分区：
文献类型：
--
作者：
N. Thompson;D. Hager;R. Burgess
使用改进的酶联免疫吸附测定 (ELISA) 来筛选与大肠杆菌 RNA 聚合酶反应的单克隆抗体 (MAb)，以确定其在低分子量多羟基化合物（多元醇）和非离液盐存在下释放 RNA 聚合酶的能力。该测定被称为 ELISA 洗脱测定，从总共筛选的 218 种抗原特异性 MAb 中鉴定出 19 种假定的“多元醇反应性”MAb。其中一种单克隆抗体被命名为 NT73，详细检查了其响应多元醇和盐的各种组合而释放抗原的能力。使用与 Sepharose 缀合的 NT73，可以通过单个免疫亲和层析步骤快速制备高活性 RNA 聚合酶，取代了我们传统纯化程序中的两个冗长的层析步骤。由于 NT73 与 RNA 聚合酶的 β' 亚基反应，因此从免疫亲和柱中回收了核心聚合酶和全酶的混合物。通过随后在 Mono Q 柱上的层析，可以将全酶（E sigma 70）与核心聚合酶分离。这表明多元醇反应性 MAb 可以通过 ELISA 洗脱测定轻松鉴定和表征。多元醇响应性单克隆抗体的使用提供了一种采用免疫亲和层析来纯化不稳定蛋白质的方法。
A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.