Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.
cell-surface engineering
site-specific labeling
substrate mimetics
trypsiligase
trypsin variants
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
06 04 2021
06 04 2021
Historique:
revised:
10
11
2020
received:
15
10
2020
pubmed:
12
11
2020
medline:
18
11
2021
entrez:
11
11
2020
Statut:
ppublish
Résumé
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 N 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 N 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.
Identifiants
pubmed: 33174659
doi: 10.1002/cbic.202000718
pmc: PMC8048679
doi:
Substances chimiques
Dipeptides
0
Fluorescent Dyes
0
enhanced green fluorescent protein
0
Basigin
136894-56-9
Green Fluorescent Proteins
147336-22-9
ErbB Receptors
EC 2.7.10.1
Trypsin
EC 3.4.21.4
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1201-1204Subventions
Organisme : SPP 1623 "Chemoselctive reactions for the synthesis and application of functional proteins"
Informations de copyright
© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.
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