Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement. (4th August 2016)

• Record Type: Journal Article
• Authors: Kallemeijn, Wouter W.; Scheij, Saskia; Voorn‐Brouwer, Tineke M.; Witte, Martin D.; Verhoek, Marri; Overkleeft, Hermen S.; Boot, Rolf G.; Aerts, Johannes M. F. G.
• Is Part Of: Chembiochem. Volume 17:Number 18(2016)
• Page Start: 1698
• Page End: 1704
• Publication Date: 2016-08-04
• Subjects: activity-based probes -- EGCaseII -- fluorescent probes -- in-gel imaging -- microscopy -- protein expression; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201600312
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : A dual‐action tag . Appending proteins with Rhodococcus sp. endo ‐β‐glucosidase (EGCaseII) enables sensitive, specific, and dual detection of fusion proteins in vitro and within their cellular context, by exploiting the tag's ability to hydrolyze 4MU‐β‐d ‐Lac (an activity absent in mammalian cells), and by mechanism‐based labeling with probes equipped with fluorophores or biotin epitopes.