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Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution
Wang, Xueying1,2; Wang, Lei1; Lin, Xinping1; Yang, Xiaobing1; Liu, Wujun1; Zhao, Zongbao K.1,3
KeywordDirected Evolution Fusion Protein Library Screening Monomeric Red Fluorescent Protein Soluble Protein Reporter
Source PublicationAPPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
2018-05-01
ISSN0273-2289
DOI10.1007/s12010-017-2640-z
Volume185Issue:1Pages:81-90
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS KeywordENHANCING FUNCTIONAL EXPRESSION ; ESCHERICHIA-COLI ; SOLUBILITY ; ENZYME ; LIBRARY
AbstractDirected evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within the nadD gene, used to transform Escherichia coli DH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution.
Language英语
WOS IDWOS:000431499100007
PublisherHUMANA PRESS INC
Citation statistics
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/167211
Collection中国科学院大连化学物理研究所
Corresponding AuthorZhao, Zongbao K.
Affiliation1.Chinese Acad Sci, Div Biotechnol, Dalian Inst Chem Phys, Dalian 116023, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Chinese Acad Sci, Dalian Natl Lab Clean Energy, Dalian Inst Chem Phys, Dalian 116023, Peoples R China
Recommended Citation
GB/T 7714
Wang, Xueying,Wang, Lei,Lin, Xinping,et al. Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution[J]. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,2018,185(1):81-90.
APA Wang, Xueying,Wang, Lei,Lin, Xinping,Yang, Xiaobing,Liu, Wujun,&Zhao, Zongbao K..(2018).Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution.APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,185(1),81-90.
MLA Wang, Xueying,et al."Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution".APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 185.1(2018):81-90.
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