Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b
Single-step immunoaffinity purification and functional reconstitution of human phagocyte flavocytochrome b
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DOI：
10.1016/j.jim.2007.10.008
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发表时间：
2008-01-01
影响因子：
2.2
通讯作者：
Taylor, Ross M.
中科院分区：
文献类型：
--
作者：
Lord, Connie I.;Riesselman, Marcia H.;Taylor, Ross M.
人中性粒细胞黄细胞色素B（Cyt B）是一种异源二聚体膜蛋白，在多亚基NADPH氧化酶复合物中产生高水平的超氧化物。由于Cyt B目前分离的量有限，因此从低水平的起始膜（来自嗜中性粒细胞和其他表达细胞类型）纯化的改进方法对于结构和催化机制的分析是重要的。在本研究中，表位定位的单克隆抗体CS 9被耦合到琼脂糖凝胶珠，并用作一步免疫亲和纯化的细胞色素B的亲和基质。在非离子去污剂辛基葡糖苷中溶解人嗜中性粒细胞和PL B-985膜级分后，将Cyt B吸附在CS 9-琼脂糖亲和基质上，并在非变性条件下用表位模拟肽洗脱纯化的蛋白质。该分离程序的高效率使得Cyt B能够从容易获得的起始膜级分水平（9 × 10（8）细胞当量的中性粒细胞膜和2 × 10（9）细胞当量的PL B-985膜）中重复纯化。由于Cyt B可以在洗涤剂辛基葡糖苷中亲和纯化，因此通过简单添加溶解的磷脂和随后的透析以除去洗涤剂，直接在洗脱级分上进行高水平的功能性重构。据我们所知，这项研究描述了最有效的方法，用于产生纯化的，功能重建的细胞色素B，并应有助于分析，需要一个高度定义的NADPH氧化酶系统。(C)2007 Elsevier B. V.保留所有权利。
Human neutrophil flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that generates high levels of superoxide in the multisubunit NADPH oxidase complex. Since Cyt b is currently isolated in limited quantities, improved methods for purification from low levels of starting membranes (from both neutrophils and other expressing cell types) are important for the analysis of structure and catalytic mechanism. In the present study, the epitope-mapped monoclonal antibody CS9 was coupled to Sepharose beads and used as an affinity matrix for single-step immunoaffinity purification of Cyt b. Following solubilization of both human neutrophil and PLB-985 membrane fractions in the nonionic detergent octylglucoside, Cyt b was absorbed on the CS9-Sepharose affinity matrix and purified protein was eluted under non-denaturing conditions with an epitope-mimicking peptide. The high efficiency of this isolation procedure allowed Cyt b to be reproducibly purified from readily obtainable levels of starting membrane fractions (9 x 10(8) cell equivalents of neutrophil membranes and 2 x 10(9) cell equivalents of PLB-985 membranes). Since Cyt b could be affinity-purified in the detergent octylglucoside, high-level functional reconstitution was carried out directly on elution fractions by simple addition of solubilized phospholipid and subsequent dialysis for detergent removal. To our knowledge, this study describes the most efficient method for generating purified, functionally-reconstituted Cyt b and should facilitate analyses that require a highly-defined NADPH oxidase system. (C) 2007 Elsevier B.V. All rights reserved.