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Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement
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.
- All authors
- Kallemeijn, W.W.; Scheij, S.; Voorn-Brouwer, T.M.; Witte, M.D.; Verhoek, M.; Overkleeft, H.S.; Boot, R.G.; Aerts, J.M.
- Date
- 2016-09-15
- Volume
- 17
- Issue
- 18
- Pages
- 1698 - 1704