A Versatile and Efficient High-Throughput Cloning Tool for Structural Biology
A Versatile and Efficient High-Throughput Cloning Tool for Structural Biology
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DOI：
10.1021/bi200178z
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发表时间：
2011-04-19
期刊：
影响因子：
2.9
通讯作者：
Dutzler, Raimund
中科院分区：
文献类型：
--
作者：
Geertsma, Eric R.;Dutzler, Raimund
将大量开放阅读框克隆到表达载体中的方法对于具有挑战性的结构生物学项目至关重要。在这里，我们描述了一个系统，称为片段交换（FX）克隆，有利于高通量的表达构建体的产生。该方法基于US类限制性内切酶和阴性选择标记。FX克隆结合了已建立的不依赖重组和连接的克隆方法的吸引人的特征：它允许将开放阅读框直接转移到各种表达载体中，并且在其使用中是高效且非常经济的。此外，FX克隆避免了用克隆相关序列显著延伸靶开放阅读框的常见但不期望的特征，因为它在蛋白质的任一侧留下仅单个额外氨基酸的最小接缝。该方法已被证明是非常强大的，适用于所有常见的原核和真核表达系统。与传统方法相比，它大大加快了表达构建体的生成，从而有利于更广泛的表达筛选。
Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class US restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.