一种改进的基于PCR的建立饱和突变库的方法  

An improved PCR-based method to create saturated mutagenic library

作　　者：佘文文 倪静 马立新[1] SHE Wenwen;NI Jing;MA Lixin(College of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2019年第1期1-4,9,共5页Journal of Hubei University：Natural Science

基　　金：湖北省技术创新重大项目(2017ACA174)资助

摘　　要：定点饱和诱变技术一直以来被广泛应用于蛋白的定向进化和代谢途径的研究,然而建立偏爱性低的高质量饱和突变库一直面临着严峻挑战.提出一种改进的建立智能突变体文库的方法,选取柠檬烯环氧水解酶(LEH)为突变对象,摒弃传统的NNK/NNN密码子简并引物,采用半理性设计突变氨基酸的方法,将PCR反扩载体与T5介导的克隆方法联用,构建一个随机的四点组合突变体库,突变效率高达81. 25%.此外,突变库的偏爱性大大降低,突变氨基酸的分布趋于平均,表现出低测序倍数基础上的高覆盖率.因此该方法极大提高了突变库的质量,降低了筛选成本,为蛋白质工程和生物合成的研究提供了好的思路.Although saturation mutagenesis(SM)technique has been widely used in the directed evolution and research on metabolic paths,creating a library with high efficiency and low bias has been challenging.In this paper,we present an improved method to construct mutagenic library,limonene epoxide hydrolase(LEH)was chosen to be mutated,instead of employing the traditional NNK/NNN codon degeneracy as building blocks,rationally chosen reduced amino acid alphabets were introduced by combination of PCR reaction and T5 exonuclease,a comprehensive combinatorial library on four randomization sites of LEH was successfully constructed,81.25%of the theoretically possible DNA mutants are obtained,which increased by 10%compared with traditional method.Besides,the percentage distribution of amino acids at each position were balanced,which showed low bias and a high coverage of programmed mutants with low sequence fold.Therefore,our method revealed great potential in protein engineering and synthetic biology by enhancing the quality of the libraries and reducing the screening cost.

关 键 词：定点突变 饱和突变 PCR T5核酸外切酶 偏爱性 

分 类 号：Q819[生物学—生物工程]