title: Controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid.
abstract: Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis of Rab1 binding onto the DrrA-immobilized surfaces using surface plasmon resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis.
authors.forename: Moon-Hyeong
authors.initials: MH
authors.lastname: Seo
authors.forename: Jieun
authors.initials: J
authors.lastname: Han
authors.forename: Zongwen
authors.initials: Z
authors.lastname: Jin
authors.forename: Dong-Won
authors.initials: DW
authors.lastname: Lee
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Park
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D057930:Click Chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D016952:Legionella pneumophila / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D020761:rab1 GTP-Binding Proteins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C101895:DrrA protein, Bacteria; D020761:rab1 GTP-Binding Proteins