Ligation independent cloning vectors for expression of SUMO fusions
Ligation independent cloning vectors for expression of SUMO fusions
复制标题
DOI：
10.1016/j.pep.2006.12.006
复制
发表时间：
2007-05-01
影响因子：
1.6
通讯作者：
Loll, Patrick J.
中科院分区：
文献类型：
--
作者：
Weeks, Stephen D.;Drinker, Mark;Loll, Patrick J.
随着对用于生物物理或生物化学分析的许多不同蛋白质的生产的需求增加，需要用于天然重组蛋白质的克隆、表达和纯化的快速方法。特别地，需要不依赖于靶基因序列的通用方法。为了解决这一挑战，我们构建了四种大肠杆菌表达载体，其可用于扩增的靶基因序列的连接非依赖性克隆（LIC）。这些载体代表具有两个不同亲和标签的两个不同亲本载体骨架的组合配对。将靶基因克隆到编码亲和力标记的小泛蛋白相关修饰剂（SUMO）的序列下游。使用增强型绿色荧光蛋白（eGFP）作为一个例子，我们证明了LIC程序的工作效率高，所有四个载体。我们还表明，所得到的重组SUMO融合蛋白可以在大肠杆菌中过表达，并容易通过标准的亲和纯化技术分离。重要的是，纯化的融合产物可以用重组SUMO水解酶处理，以产生在氨基末端具有除脯氨酸之外的任何残基的成熟靶蛋白。我们通过产生含有非天然氨基末端半胱氨酸残基的重组eGFP并将其用作表达蛋白连接（EPL）的底物来证明这一点的应用。本文所述的试剂和技术代表了用于快速克隆和生产靶蛋白的通用方法，并且适用于高通量基因组规模表达项目。(c)2006年爱思唯尔公司All rights reserved.
With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project. (c) 2006 Elsevier Inc. All rights reserved.