| RRC ID |
77275
|
| 著者 |
Yamaguchi A, Matsuda T, Ohtake K, Yanagisawa T, Yokoyama S, Fujiwara Y, Watanabe T, Hohsaka T, Sakamoto K.
|
| タイトル |
Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates.
|
| ジャーナル |
Bioconjug Chem
|
| 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.
|
| 巻・号 |
27(1)
|
| ページ |
198-206
|
| 公開日 |
2016-1-20
|
| DOI |
10.1021/acs.bioconjchem.5b00602
|
| PMID |
26625213
|
| MeSH |
Amino Acids / chemistry*
Antibodies / chemistry
Azides / chemistry
Biochemistry / methods*
ErbB Receptors / chemistry
ErbB Receptors / immunology
Fluorescent Dyes / chemistry
Methanosarcina / enzymology
Polyethylene Glycols / chemistry
Proteins / chemistry*
Single-Domain Antibodies / chemistry*
Surface Plasmon Resonance
|
| IF |
4.031
|
| リソース情報 |
| 遺伝子材料 |
pCDF-Az (RDB20115)
pCDF-Mm2 (RDB20116) |
