title: Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates.
abstract: Z-Lysine (ZLys) is a lysine derivative with a benzyloxycarbonyl group linked to the ε-nitrogen. It has been genetically encoded with the UAG stop codon, using the pair of an engineered variant of pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl). In the present study, we designed a novel Z-lysine derivative (AmAzZLys), which is doubly functionalized with amino and azido substituents at the meta positions of the benzyl moiety, and demonstrated its applicability for creating protein conjugates. AmAzZLys was incorporated into proteins in Escherichia coli, by using the ZLys-specific PylRS variant. AmAzZLys was then site-specifically incorporated into a camelid single-domain antibody specific to the epidermal growth factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl amine and azide were efficiently linked to the 5 kDa polyethylene glycol and a fluorescent probe, respectively, through specific bio-orthogonal chemistry. The antibody was then tested for the ability to form a photo-cross-link between its phenylazide moiety and the antigen, while the amino group on the same ring was used for chemical labeling. When incorporated at a selected position in the antibody and exposed to 365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain, with the phenylamine moiety labeled fluorescently prior to the reaction. The present results illuminated the versatility of the ZLys scaffold, which can accommodate multiple reactive groups useful for protein conjugation.
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology , 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohtake
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yanagisawa
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: Shinsei Chemical Company Ltd. , 7-7-15 Saitoasagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology , 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology , 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000906:Antibodies / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D066246:ErbB Receptors / Q000737:chemistry / Q000276:immunology; D005456:Fluorescent Dyes / Q000737:chemistry; D017020:Methanosarcina / Q000201:enzymology; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D061905:Single-Domain Antibodies / Q000737:chemistry*; D020349:Surface Plasmon Resonance
chemical_list: D000596:Amino Acids; D000906:Antibodies; D001386:Azides; D005456:Fluorescent Dyes; D011506:Proteins; D061905:Single-Domain Antibodies; D011092:Polyethylene Glycols; C014747:phenylazide; C512478:EGFR protein, human; D066246:ErbB Receptors