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Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein
1Center for Molecular Biology and Gene Therapy and Department of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350, USA.
Luminescence : the Journal of Biological and Chemical Luminescence
|February 8, 2000
Summary
Researchers engineered a secreted Renilla luciferase (SRUC)-green fluorescent protein (GFP) fusion. This chimeric protein successfully visualized and quantified protein secretion in mammalian cells, demonstrating its utility as a novel marker.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Engineered Renilla luciferase (SRUC) is secreted as a functional enzyme by mammalian cells.
- Fusing wild-type Renilla luciferase with green fluorescent protein (GFP) creates a chimeric protein with retained luminescence properties.
Purpose of the Study:
- To determine if SRUC fused with GFP can visualize and quantify protein secretion in mammalian cells.
- To assess the functionality of the secreted SRUC-GFP fusion protein.
Main Methods:
- Transient transfection of Simian COS-7 and Chinese hamster ovary (CHO) cells with gene constructs for secreted or intracellular SRUC-GFP fusion proteins.
- Measurement of Renilla luciferase activity in cell lysates and culture media.
- Detection of GFP activity in CHO cells using fluorescence microscopy.
Main Results:
- The SRUC-GFP fusion protein was successfully secreted from mammalian cells as a chimeric protein.
- The secreted fusion protein retained both Renilla luciferase and GFP activity.
- Data confirmed the dual functionality of the secreted SRUC-GFP fusion protein.
Conclusions:
- The SRUC-GFP fusion protein is a viable and functional marker for studying protein secretion in mammalian cells.
- This chimeric protein offers a dual-mode system for both visualization and quantification of secreted proteins.