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Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation

Ramasamy Paulmurugan¹, Sanjiv S Gambhir

¹Departments of Radiology and Bioengineering, Bio-X Program, Molecular Imaging Program at Stanford, Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, Stanford, California 94305-5427, USA. paulmur8@stanford.edu

Analytical Chemistry

|February 14, 2007

Summary

Researchers developed a novel split firefly luciferase system for enhanced bioluminescence imaging. This new system, Nfluc 398/Cfluc 394, offers superior signal and lower background for studying protein interactions in cells and living animals.

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Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Split reporter-based bioluminescence imaging is crucial for studying intracellular interactions.
Existing split sites for firefly luciferase have limitations in signal and background noise.

Purpose of the Study:

To identify and characterize a novel split site for firefly luciferase with improved imaging capabilities.
To validate the new split site's performance in various biological contexts.

Main Methods:

A combinatorial strategy was employed to screen 115 split site combinations.
Characterization involved five interacting protein partners and intramolecular folding studies.
Validation included cell culture imaging and in vivo imaging in living mice.

Main Results:

A novel split site, Nfluc 398/Cfluc 394, was identified with a greater absolute signal and near-zero background.
This new combination demonstrated markedly superior performance compared to existing split luciferase fragments.
The Nfluc 398/Cfluc 394 system exhibited lower self-complementation and equal or higher post-interaction signal.

Conclusions:

The Nfluc 398/Cfluc 394 split luciferase system offers significant advantages for protein-protein interaction studies.
This novel system can be applied to diverse cellular events, including subcellular localization and cell fusion.
The improved system enhances bioluminescence imaging applications in both cellular and in vivo models.