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

作者：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^*

来源：ChemBioChem, 2016, 17(18): 1698-1704.

DOI：10.1002/cbic.201600312

• activity-based probes
• EGCaseII
• fluorescent probes
• in-gel imaging
• microscopy
• protein expression

beta-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of -glucosidases. Their applicability to detect proteins fused with -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 -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 -epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous -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.

• 出版日期2016-9-15