An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A

doi:10.1016/j.bbrc.2017.03.016

	An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A
	Li, Ning1,2,3 ; Yu, Zheng1,2,3 ; Ji, Qun1 ; Sun, Jingying1,2,3 ; Liu, Xiao1,2,3 ; Du, Mingjuan1 ; Zhang, Wei1
	2017-04-29
发表期刊	BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (IF:2.5[JCR-2023],2.7[5-Year])
ISSN	0006-291X
卷号	486 期号:2 页码:257-263
发表状态	已发表
DOI	10.1016/j.bbrc.2017.03.016
摘要	Enzyme-mediated protein conjugation has gained great attention recently due to the remarkable site selectivity and mild reaction condition affected by the nature of enzyme. Among all sorts of enzymes reported, sortase A from Staphylococcus aureus (SaSrtA) is the most popular enzyme due to its selectivity and well-demonstrated applications. Position scanning has been widely applied to understand enzyme substrate specificity, but the low throughput of chemical synthesis of peptide substrates and analytical methods (HPLC, LC-ESI-MS) have been the major hurdle to fully decode enzyme substrate profile. We have developed. a simple high-throughput substrate profiling method to reveal novel substrates of SaSrtA 7M, a widely used hyperactive peptide ligase, by modified protein-fragment complementation assay (PCA). A small library targeting the LPATG motif recognized by SaSrtA 7M was generated and screened against proteins carrying N-terminal glycine. Using this method, we have confirmed all currently known substrates of the enzyme, and moreover identified some previously unknown substrates with varying activities. The method provides an easy, fast and highly-sensitive way to determine substrate profile of a peptide ligase in a high-throughput manner. (C) 2017 Elsevier Inc. All rights reserved.
关键词	High-throughput screening NanoLuc luciferase Protein-fragment complementation assay Sortase A Substrate profiling
收录类别	SCI
语种	英语
资助项目	National Natural Science Foundation of China[31600628]
WOS研究方向	Biochemistry & Molecular Biology ; Biophysics
WOS类目	Biochemistry & Molecular Biology ; Biophysics
WOS记录号	WOS:000399261200008
出版者	ACADEMIC PRESS INC ELSEVIER SCIENCE
WOS关键词	STAPHYLOCOCCUS-AUREUS SORTASE ; BIMOLECULAR FLUORESCENCE COMPLEMENTATION ; LIVING CELLS ; TRANSPEPTIDASE-SRTA ; ESCHERICHIA-COLI ; SURFACE-PROTEINS ; IN-VIVO ; VISUALIZATION ; SPECIFICITY ; EFFICIENCY
原始文献类型	Article
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/1402
专题	生命科学与技术学院免疫化学研究所_特聘教授组_功能筛选实验室免疫化学研究所_特聘教授组_抗体设计学实验室生命科学与技术学院_硕士生
通讯作者	Zhang, Wei
作者单位	1.ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China 2.Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, Shanghai 200031, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
第一作者单位	免疫化学研究所
通讯作者单位	免疫化学研究所
第一作者的第一单位	免疫化学研究所
推荐引用方式 GB/T 7714	Li, Ning,Yu, Zheng,Ji, Qun,et al. An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A[J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,2017,486(2):257-263.
APA	Li, Ning.,Yu, Zheng.,Ji, Qun.,Sun, Jingying.,Liu, Xiao.,...&Zhang, Wei.(2017).An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,486(2),257-263.
MLA	Li, Ning,et al."An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A".BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 486.2(2017):257-263.