Expression and purification of SARS coronavirus proteins using SUMO-fusions.
Expression and purification of SARS coronavirus proteins using SUMO-fusions.
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DOI：
10.1016/j.pep.2005.02.004
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发表时间：
2005-07
影响因子：
1.6
通讯作者：
Butt TR
中科院分区：
文献类型：
--
作者：
Zuo X;Mattern MR;Tan R;Li S;Hall J;Sterner DE;Shoo J;Tran H;Lim P;Sarafianos SG;Kazi L;Navas-Martin S;Weiss SR;Butt TR
严重急性呼吸综合征冠状病毒（SARS-CoV）蛋白是一大类在传统表达系统中难以表达的蛋白。大量表达和纯化SARS-CoV蛋白的能力对于基础研究和药物制剂的开发至关重要。本文的研究结果表明：（1）SUMO（small ubiquitin-related modifiers，一种100个氨基酸的多肽）与SARS-CoV蛋白的N-末端的融合显著增强了SARS-CoV蛋白在大肠杆菌细胞中的表达;（2）6× His标记的SUMO融合蛋白有助于大规模快速纯化病毒蛋白。我们利用SUMO的天然伴侣特性开发了一种适用于传统方法无法表达的蛋白质的表达系统。该系统的一个独特特征是SUMO标签，其增强表达，促进纯化，并且可以被SUMO特异性蛋白酶有效切割以产生具有所需N-末端的天然蛋白质。我们已经在天然或变性条件下纯化了各种SARS-CoV蛋白。这些纯化的蛋白质已用于产生高度特异性的多克隆抗体。我们的研究表明，SUMO融合技术将有助于提高表达和纯化的病毒蛋白的结构和功能的研究，以及用于治疗用途。
Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6× His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.
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