title: Site-selective protein immobilization by covalent modification of GST fusion proteins.
abstract: The immobilization of functional proteins onto solid supports using affinity tags is an attractive approach in recent development of protein microarray technologies. Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein-protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization. Here, by utilizing pyrimidine-based small-molecule probes with a sulfonyl fluoride reactive group, we report a novel and general approach for site-selective immobilization of Schistosoma japonicum GST (sjGST) fusion proteins through irreversible and specific covalent modification of the tyrosine-111 residue of the sjGST tag. As demonstrated by sjGST-tagged eGFP and sjGST-tagged kinase activity assays, this immobilization approach offers the advantages of high immobilization efficiency and excellent retention of protein structure and activity.
authors.affiliation: Key Laboratory of Chemical Genomics, Key Laboratory of Structural Biology, School of Chemical Biology and Biotechnology, Peking University, Shenzhen Graduate School , Xili University Town, PKU Campus, Shenzhen 518055, China.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Tianlin
authors.initials: T
authors.lastname: Guo
authors.forename: Guanghui
authors.initials: G
authors.lastname: Tang
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.forename: Nai-Kei
authors.initials: NK
authors.lastname: Wong
authors.forename: Zhengying
authors.initials: Z
authors.lastname: Pan
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012549:Schistosoma japonicum; D013379:Substrate Specificity
chemical_list: D000480:Alkynes; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins