Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers
Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers
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DOI：
10.2144/000113820
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发表时间：
2012-03-01
期刊：
影响因子：
2.7
通讯作者：
Jiang, Rongxiang
中科院分区：
文献类型：
--
作者：
Tang, Lixia;Gao, Hui;Jiang, Rongxiang
位点饱和诱变技术是一种高效、简便的蛋白质优化方法。通常使用简并密码子NNN或NNS（K）来编码20个标准氨基酸，但这会产生冗余密码子，并且导致构建的文库中氨基酸分布不均匀。在这里，我们提出了一种新的“小智能”的策略来构建诱变文库，具有最小的基因库的大小没有固有的氨基酸偏差，终止密码子，或罕见的密码子大肠杆菌通过耦合精心设计的组合简并引物与合适的PCR为基础的诱变方法。所设计的引物混合物包含每个氨基酸恰好一个密码子，因此允许构建每个蛋白质一个基因的小型智能诱变文库。此外，开发了软件工具DC-Analyzer，以根据用户定义的文库构建随机化方案辅助引物设计。这种小智能策略被成功地应用于卤代醇脱卤酶的随机化与一个或两个随机化的网站。在DC-Analyzer的帮助下，该策略被证明与NNS随机化一样简单，并且可以作为一种通用工具来有效地随机化感兴趣位置的靶基因。
Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel "small-intelligent" strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.