Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement

Wouter W. Kallemeijn, Saskia Scheij, Tineke M. Voorn-Brouwer, Martin D. Witte, Marri Verhoek, Hermen S. Overkleeft, Rolf G. Boot, Johannes M. F. G. Aerts

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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
Original languageEnglish
Pages (from-to)1698-1704
JournalChembiochem
Volume17
Issue number18
DOIs
Publication statusPublished - 2016

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