Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction
Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction
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DOI：
10.1002/jmr.926
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发表时间：
2009-03-01
影响因子：
2.7
通讯作者：
Bayer, Edward A.
中科院分区：
文献类型：
--
作者：
Karpol, Alon;Kantorovich, Lia;Bayer, Edward A.
厌氧嗜热细菌热纤梭菌对纤维素的有效降解是通过多酶纤维素体复合物进行的。复合物上的酶通过其 dockerin (Doc) 模块以钙依赖性方式连接到纤维素体支架蛋白亚基的粘连蛋白 (Coh) 模块。在本研究中，我们优化了 Coh-Doc 相互作用，以实现蛋白质亲和纯化的目的。因此，热纤梭菌Coh模块与碳水化合物结合模块融合，并将所得融合蛋白直接施加到珠状纤维素上，从而充当非共价“激活”程序。然后将互补的 Doc 模块融合到模型蛋白靶标：来自嗜热脂肪芽孢杆菌的木聚糖酶 T-6。然而，在用 EDTA 处理后，与固定化 Coh 的结合仅部分可逆，并且仅从亲和柱中洗脱了可忽略量的靶蛋白。为了改善蛋白质洗脱，设计了一系列截短的Doc，其中钙协调功能受损，但不明显影响与Coh的高亲和力结合。仅 48 个残基的缩短 Doc 足以充当有效的亲和标签，并且只需一步即可直接从粗细胞提取物中获得高度纯化的目标蛋白，并且对目标蛋白进行近乎定量的回收。有效的 EDTA 介导的从柱中洗脱隔离的蛋白质是该过程的关键步骤。亲和柱可重复使用，并且在重复加载和洗脱轮次后仍保持非常高水平的容量。因此，可重复使用的 Coh-Doc 亲和柱通过修改 Doc 模块的钙结合环，提供了一种高效且有吸引力的高产率纯化蛋白质的方法。版权所有 (c) 2008 John Wiley & Sons, Ltd.
Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the mufti-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. Copyright (c) 2008 John Wiley & Sons, Ltd.