Introducing inducible fluorescent split cholesterol oxidase to mammalian cells

Konstantin G. Chernov, Maarit Neuvonen, Ivonne Brock, Elina Ikonen, Vladislav Verkhusha

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 from Chromobacterium sp. 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 GFPCOase, 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.

Original languageEnglish (US)
Pages (from-to)8811-8822
Number of pages12
JournalJournal of Biological Chemistry
Volume292
Issue number21
DOIs
StatePublished - May 26 2017

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Cholesterol Oxidase
Cholesterol
Cells
Sirolimus
Enzymes
Fluorescence
Chromobacterium
Tacrolimus Binding Proteins
Biosensing Techniques
Cell membranes
Insecticides
Biodegradation
Biosensors
Steroids
Cell Membrane
Oxidation
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells. / Chernov, Konstantin G.; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav.

In: Journal of Biological Chemistry, Vol. 292, No. 21, 26.05.2017, p. 8811-8822.

Research output: Contribution to journalArticle

Chernov, Konstantin G. ; Neuvonen, Maarit ; Brock, Ivonne ; Ikonen, Elina ; Verkhusha, Vladislav. / Introducing inducible fluorescent split cholesterol oxidase to mammalian cells. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 21. pp. 8811-8822.
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