title: Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.
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.
authors.affiliation: Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL.
authors.forename: Wouter W
authors.initials: WW
authors.lastname: Kallemeijn
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, NL.
authors.forename: Saskia
authors.initials: S
authors.lastname: Scheij
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, NL.
authors.forename: Tineke M
authors.initials: TM
authors.lastname: Voorn-Brouwer
authors.affiliation: Department of Bio-Organic Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, NL.|Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Witte
authors.affiliation: Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL.
authors.forename: Marri
authors.initials: M
authors.lastname: Verhoek
authors.affiliation: Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL.
authors.forename: Hermen S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL.
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.affiliation: Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, NL. j.m.f.g.aerts@lic.leidenuniv.nl.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008361:Mannosidases / Q000378:metabolism*; D015394:Molecular Structure; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology
chemical_list: D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D008361:Mannosidases; C119276:endo-beta-mannosidase
keywords: EGCaseII; activity-based probes; fluorescent probes; in-gel imaging; microscopy; protein expression