Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution
文献类型：期刊论文
| 作者 | Yang, Xiaobing2; Zhao, Zongbao K.2,3; Wang, Xueying1,2; Wang, Lei2; Lin, Xinping2; Liu, Wujun2 |
| 刊名 | APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
 |
| 出版日期 | 2018-05-01 |
| 卷号 | 185期号:1页码:81-90 |
| 关键词 | Directed Evolution Fusion Protein Library Screening Monomeric Red Fluorescent Protein Soluble Protein Reporter |
| ISSN号 | 0273-2289 |
| DOI | 10.1007/s12010-017-2640-z |
| 文献子类 | Article |
| 英文摘要 | Directed 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. |
| WOS关键词 | ENHANCING FUNCTIONAL EXPRESSION ; ESCHERICHIA-COLI ; SOLUBILITY ; ENZYME ; LIBRARY |
| WOS研究方向 | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 语种 | 英语 |
| WOS记录号 | WOS:000431499100007 |
| 出版者 | HUMANA PRESS INC |
| 源URL | [http://cas-ir.dicp.ac.cn/handle/321008/167210]  |
| 专题 | 大连化学物理研究所_中国科学院大连化学物理研究所 |
| 通讯作者 | Zhao, Zongbao K. |
| 作者单位 | 1.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 2.Chinese Acad Sci, Div Biotechnol, Dalian Inst Chem Phys, Dalian 116023, Peoples R China 3.Chinese Acad Sci, Dalian Natl Lab Clean Energy, Dalian Inst Chem Phys, Dalian 116023, Peoples R China |
| 推荐引用方式 GB/T 7714 | Yang, Xiaobing,Zhao, Zongbao K.,Wang, Xueying,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 | Yang, Xiaobing,Zhao, Zongbao K.,Wang, Xueying,Wang, Lei,Lin, Xinping,&Liu, Wujun.(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 | Yang, Xiaobing,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. |
入库方式： OAI收割
来源：大连化学物理研究所
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