An improved SUMO fusion protein system for effective production of native proteins
An improved SUMO fusion protein system for effective production of native proteins
复制标题
DOI：
10.1110/ps.035188.108
复制
发表时间：
2008-07-01
期刊：
影响因子：
8
通讯作者：
Wang, Ting-Fang
中科院分区：
文献类型：
--
作者：
Lee, Chien-Der;Sun, Hui-Chien;Wang, Ting-Fang
重组蛋白与 SUMO（小泛素相关修饰剂）蛋白融合的表达显着提高了大肠杆菌中难以表达的蛋白的产量。天然存在的 SUMO 蛋白酶可从融合蛋白中去除 SUMO，进一步增强了该技术的优势。在这里，我们改进了现有的 SUMO 融合蛋白方法，以有效生产天然蛋白。首先，应用粘端PCR策略设计一种新的SUMO融合蛋白载体，该载体允许使用两个通用克隆位点（5'端的Sfol和3'端的XhoI）定向克隆任何靶基因。目标基因 PCR 产物不需要限制性酶切，即使插入的目标基因含有 SfoI 或 XhoI 限制性位点。该载体产生融合蛋白（表示为 His(6)-Smt3-X），其中目标蛋白 (X) 与六组氨酸 (His(6)) 标记的 Smt3 融合。 Smt3 是酵母 SUMO 蛋白。 His(6)-Smt(3)-X经Ni2+树脂纯化。通过工程SUMO蛋白酶His(6)-Ulpl(403-621)-His(6)在Ni2+树脂上去除His(6)-Smt3。由于其双His(6)标签，His(6)-Ulpl(403-621)-His(6)表现出对Ni 2+ 树脂的高亲和力，并在裂解反应后与Ni 2+ 树脂缔合。使用一根 Ni2+-树脂柱即可进行融合蛋白纯化和 SUMO 蛋白酶裂解。洗脱液仅含有天然目标蛋白。这种单列协议对于开发更好的高通量平台很有用。最后，这个新系统被证明可以有效地克隆、表达和快速纯化几种难以生产的真实蛋白质。
Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfol at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt(3)-X was purified by Ni2+ resin. Removal of His(6)- Smt3 was performed on the Ni2+ resin by an engineered SUMO protease, His(6)-Ulpl(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulpl(403-621)-His(6) exhibits a high affinity for Ni2+ resin and associates with Ni2+ resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni2+-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.