DICP OpenIR
PCR-based strategy for construction of multi-site-saturation mutagenic expression library
Wang, Jinxia1,2; Zhang, Sufang1; Tan, Haidong1; Zhao, Zongbao (Kent)1
KeywordExpression Library Pcr Cloning Protein Engineering Site-saturation Mutagenesis
Source PublicationJOURNAL OF MICROBIOLOGICAL METHODS
2007-12-01
DOI10.1016/j.mimet.2007.09.001
Volume71Issue:3Pages:225-230
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
WOS SubjectBiochemical Research Methods ; Microbiology
WOS Research AreaBiochemistry & Molecular Biology ; Microbiology
WOS KeywordNAD-MALIC ENZYME ; DIRECTED EVOLUTION ; CLONING ; DESIGN
AbstractThere 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.
Language英语
WOS IDWOS:000252080300007
Citation statistics
Cited Times:12[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/140594
Collection中国科学院大连化学物理研究所
Affiliation1.Dalian Inst Chem Phys, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
Recommended Citation
GB/T 7714
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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