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PCR-based strategy for construction of multi-site-saturation mutagenic expression library
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
2007-12-01
DOI10.1016/j.mimet.2007.09.001
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
关键词[WOS]NAD-MALIC ENZYME ; DIRECTED EVOLUTION ; CLONING ; DESIGN
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.
语种英语
WOS记录号WOS:000252080300007
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被引频次:12[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/140594
专题中国科学院大连化学物理研究所
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
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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.
APA Wang, Jinxia,Zhang, Sufang,Tan, Haidong,&Zhao, Zongbao .(2007).PCR-based strategy for construction of multi-site-saturation mutagenic expression library.JOURNAL OF MICROBIOLOGICAL METHODS,71(3),225-230.
MLA Wang, Jinxia,et al."PCR-based strategy for construction of multi-site-saturation mutagenic expression library".JOURNAL OF MICROBIOLOGICAL METHODS 71.3(2007):225-230.
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