An optimized system for expression and purification of secreted bacterial proteins
An optimized system for expression and purification of secreted bacterial proteins
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DOI：
10.1016/j.pep.2005.09.003
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发表时间：
2006-03-01
影响因子：
1.6
通讯作者：
Pop, M
中科院分区：
文献类型：
--
作者：
Geisbrecht, BV;Bouyain, S;Pop, M
在这份报告中，我们描述了一个优化的系统，有效的过表达，纯化和重折叠的分泌细菌蛋白。候选分泌蛋白在大肠杆菌中重组产生为烟草蚀纹病毒蛋白酶可切割的六组氨酸-cinyc表位融合蛋白。不考虑它们的初始溶解度，用氯化胍从全细胞中提取重组融合蛋白，在变性条件下通过固定化金属aflinity色谱法纯化。并通过快速稀释到仅含Tris缓冲液和氯化钠的溶液中进行重折叠。在天然条件下在相同树脂上浓缩后，洗脱每种蛋白质用于进一步纯化和/或表征。对12种大小为13至130 kDa的分泌蛋白的测试组的初步研究产生了每升诱导培养物10至50 mg的融合蛋白，纯度大于90%，如考马斯染色的SDS-PAGE所判断的。进一步纯化的9种蛋白质，分析凝胶过滤色谱法表明，每一个是在溶液中的单体和圆二色谱显示，每个已通过了明确的二级结构。虽然该系统有许多潜在的应用，但本文提出的结果表明，它对采用结构方法来理解蛋白质功能的研究人员特别有用，如使用该方法纯化的三种蛋白质的晶体结构所证明的（B. V. Geisbrecht，B.Y. Harnaoka，B. Perman，A. Zerrila，D.J. Leally，J. Biol. Clieni. 280（2005）17243-17250）。(c)2005年爱思唯尔公司All rights reserved.
In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-cinyc eptiope fusion proteins. Without regard to their initial Solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal aflinity chromatography. and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary Studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced Culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroisin spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here sugaest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal Structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Harnaoka, B. Perman, A. Zerrila, D.J. Leally, J. Biol. Clieni. 280 (2005) 17243-17250). (c) 2005 Elsevier Inc. All rights reserved.