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题名: PCR-based strategy for construction of multi-site-saturation mutagenic expression library
作者: Wang, Jinxia1, 2;  Zhang, Sufang1;  Tan, Haidong1;  Zhao, Zongbao (Kent)1
关键词: expression library ;  PCR cloning ;  protein engineering ;  site-saturation mutagenesis
刊名: JOURNAL OF MICROBIOLOGICAL METHODS
发表日期: 2007-12-01
DOI: 10.1016/j.mimet.2007.09.001
卷: 71, 期:3, 页:225-230
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods ;  Microbiology
研究领域[WOS]: Biochemistry & Molecular Biology ;  Microbiology
英文摘要: 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.
关键词[WOS]: NAD-MALIC ENZYME ;  DIRECTED EVOLUTION ;  CLONING ;  DESIGN
语种: 英语
WOS记录号: WOS:000252080300007
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/140594
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Dalian Inst Chem Phys, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China

Recommended Citation:
Wang, Jinxia,Zhang, Sufang,Tan, Haidong,et al. PCR-based strategy for construction of multi-site-saturation mutagenic expression library[J]. JOURNAL OF MICROBIOLOGICAL METHODS,2007,71(3):225-230.
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