Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner
Single-step affinity purification of recombinant proteins using the silica-binding Si-tag as a fusion partner
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DOI：
10.1016/j.pep.2009.12.009
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发表时间：
2010-05-01
影响因子：
1.6
通讯作者：
Kuroda, Akio
中科院分区：
文献类型：
--
作者：
Ikeda, Takeshi;Ninomiya, Ken-ichi;Kuroda, Akio
我们以前报道，二氧化硅结合蛋白，指定Si标签，可以用作融合标签，以在二氧化硅表面上的功能蛋白。在本研究中，我们利用硅标签对二氧化硅的强亲和性，发展了一种一步纯化硅标签融合蛋白的方法。我们利用未改性的裸二氧化硅颗粒作为特定的吸附剂和高浓度的MgCl 2溶液作为洗脱缓冲液。从重组大肠杆菌的粗提取物中回收了Si标签和免疫球蛋白结合葡萄球菌蛋白A的融合蛋白，命名为Si标签蛋白A，纯度为87 +/- 3%，产率为84 +/- 4%。我们的方法的简单性使得能够快速、成本有效地纯化Si标记的融合蛋白。我们还讨论了Si-tag和SiO2表面结合和解离的机理，并认为Si-tag多肽的异常碱性和无序结构在SiO2与SiO2的结合中起着重要作用。(C)2009爱思唯尔公司All rights reserved.
We previously reported that a silica-binding protein, designated Si-tag, can be used as a fusion tag to immobilize functional proteins on silica surfaces. In this study, by taking advantage of the strong affinity of Si-tag for silica, we developed a single-step purification method for Si-tagged fusion proteins. We utilized unmodified bare silica particles as a specific adsorbent and a high concentration of MgCl2 solution as an elution buffer. A fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A, designated Si-tagged protein A, was recovered with a purity of 87 +/- 3% and yield of 84 +/- 4% from a crude extract of recombinant Escherichia coli. The simplicity Of Our method enables rapid, cost-effective purification of Si-tagged fusion proteins. We also discuss the mechanism of binding and dissociation of Si-tag and silica surfaces, and we suggest that the unusual basicity and disordered Structure of the Si-tag polypeptide play important roles in the binding to silica. (C) 2009 Elsevier Inc. All rights reserved.