Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.
Chembiochem
; 22(7): 1201-1204, 2021 04 06.
Article
em En
| MEDLINE
| ID: mdl-33174659
ABSTRACT
Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the Nâ
terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins' native Nâ
termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.
Palavras-chave
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Tripsina
/
Basigina
/
Receptores ErbB
/
Corantes Fluorescentes
Limite:
Humans
Idioma:
En
Revista:
Chembiochem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Alemanha