title: Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues.
abstract: To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources.
authors.affiliation: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Takamitsu
authors.affiliation: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Fukunaga
authors.affiliation: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Iio
authors.affiliation: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.
authors.forename: Koko
authors.initials: K
authors.lastname: Moriya
authors.affiliation: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. Electronic address: utsumi@yamaguchi-u.ac.jp.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Utsumi
mesh_terms: D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006801:Humans; D008567:Membranes, Artificial; D019814:Myristic Acid / Q000737:chemistry*; D011145:Polyvinyls / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D005465:Fluorocarbon Polymers
chemical_list: D004247:DNA; D008567:Membranes, Artificial; D019814:Myristic Acid; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers
keywords: Cell-free protein synthesis system; Click chemistry; Metabolic labeling; N-myristoylation