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Endo ‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement
Author(s) -
Kallemeijn Wouter W.,
Scheij Saskia,
VoornBrouwer Tineke M.,
Witte Martin D.,
Verhoek Marri,
Overkleeft Hermen S.,
Boot Rolf G.,
Aerts Johannes M. F. G.
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201600312
Subject(s) - biochemistry , fusion protein , chemistry , fluorescence , biotinylation , context (archaeology) , biology , recombinant dna , paleontology , physics , quantum mechanics , gene
β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐ d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms.