title: Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis.
abstract: The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Tokuda
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Kazushi
authors.initials: K
authors.lastname: Horiike
authors.forename: Kaori
authors.initials: K
authors.lastname: Shiraga
authors.forename: Ryoji
authors.initials: R
authors.lastname: Abe
authors.forename: Norihito
authors.initials: N
authors.lastname: Muranaka
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D049148:Aminoacylation; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D011506:Proteins; C003712:trimethyllysine; D019809:Streptavidin; D012343:RNA, Transfer; D008239:Lysine