title: Incorporation of glycosylated amino acid into protein by an in vitro translation system.
abstract: O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama 223-8522, Japan.
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Matsubara
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Iijima
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
authors.forename: Toshinori
authors.initials: T
authors.lastname: Sato
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D018388:Codon, Terminator; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D018388:Codon, Terminator; D019809:Streptavidin; D012343:RNA, Transfer; D000116:Acetylgalactosamine
keywords: Expanding genetic code; Glycoprotein; Glycosylated amino acid; In vitro translation system; O-Glycan