title: Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.
abstract: Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned fromChromobacteriumsp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells.
authors.affiliation: From the Department of Biochemistry and Developmental Biology and.
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Chernov
authors.affiliation: Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland.|Minerva Foundation Institute for Medical Research, Helsinki 00290, Finland, and.
authors.forename: Maarit
authors.initials: M
authors.lastname: Neuvonen
authors.affiliation: Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland.|Minerva Foundation Institute for Medical Research, Helsinki 00290, Finland, and.
authors.forename: Ivonne
authors.initials: I
authors.lastname: Brock
authors.affiliation: Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland, elina.ikonen@helsinki.fi.|Minerva Foundation Institute for Medical Research, Helsinki 00290, Finland, and.
authors.forename: Elina
authors.identifier: 0000-0001-8382-1135
authors.initials: E
authors.lastname: Ikonen
authors.affiliation: From the Department of Biochemistry and Developmental Biology and vladislav.verkhusha@einstein.yu.edu.|Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Vladislav V
authors.identifier: 0000-0002-2083-8121
authors.initials: VV
authors.lastname: Verkhusha
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002861:Chromobacterium / Q000201:enzymology* / Q000235:genetics; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D000072417:Protein Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000737:chemistry; D022021:Tacrolimus Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002784:Cholesterol; D002789:Cholesterol Oxidase; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus
keywords: cholesterol; cholesterol metabolism; cholesterol regulation; cholesterol-binding protein; enzyme