PCR-based strategy for construction of multi-site-saturation mutagenic expression library
PCR-based strategy for construction of multi-site-saturation mutagenic expression library
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DOI：
10.1016/j.mimet.2007.09.001
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发表时间：
2007-12-01
影响因子：
2.2
通讯作者：
Zhao, Zongbao (Kent)
中科院分区：
文献类型：
--
作者：
Wang, Jinxia;Zhang, Sufang;Zhao, Zongbao (Kent)
对于用于工业应用、结构生物学和药物开发的高效且有效的蛋白质变体工程化方法的需求日益增加。我们描述了一种基于PCR的策略，使用携带野生型基因的环状质粒产生多位点饱和诱变表达文库。这种不依赖于限制性消化和连接的方法包括三个步骤：1）合成简并寡核苷酸引物，2）通过PCR掺入突变，3）转化到表达宿主中。我们的策略是通过一个大肠杆菌K12苹果酸酶表达文库，其中包含的成员与氨基酸残基G311，D345和G397同时突变的成功构建证明。这种方法原则上与任何可以用dam（+）E传播的圆形向量兼容。coli宿主产生具有多种变化的蛋白变体文库，包括突变、短序列缺失和插入，或它们的任何混合。(c)2007 Elsevier B.V保留所有权利。
There is an increasing demand for efficient and effective methods to engineer protein variants for industrial applications, structural biology and drug development. We describe a PCR-based strategy that produces multi-site-saturation mutagenic expression library using a circular plasmid carrying the wild-type gene. This restriction digestion- and ligation-independent method involves three steps: 1) synthesis of the degenerate oligonucleotide primers, 2) incorporation of the mutations through PCR, 3) transformation into the expression host. Our strategy is demonstrated through successful construction of an E coli K12 malic enzyme expression library that contains members with simultaneous mutations on amino acid residues G311, D345 and G397. This method is in principle compatible with any circular vector that can be propagated with a dam(+) E. coli host to generate protein variant library with multiple changes, including mutation, short sequence deletion and insertion, or any mix of them. (c) 2007 Elsevier B.V All rights reserved.