title: An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A.
abstract: 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.
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Ji
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jingying
authors.initials: J
authors.lastname: Sun
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China.
authors.forename: Mingjuan
authors.initials: M
authors.lastname: Du
authors.affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China. Electronic address: zhangwei@shanghaitech.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: High-throughput screening; NanoLuc luciferase; Protein-fragment complementation assay; Sortase A; Substrate profiling