Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments

BMC Biotechnology

Yuki Ohmuro-Matsuyama²  Chan-I Chung¹  Hiroshi Ueda² 

[1] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

[2] Chemical Resources Laboratory, Tokyo Institute of Technology, R1-18, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan

关键词: In vitro diagnostics;    Thermostability;    Protein fragment complementation assay;    Bioluminescence;    Firefly luciferase;    Protein-protein interaction;   

Others  :  1123183
DOI  :  10.1186/1472-6750-13-31

 received in 2012-10-03, accepted in 2013-03-15,  发布年份 2013

【 摘 要 】

Background

Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics.

Results

Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system.

Conclusion

Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.

【 授权许可】

   
2013 Ohmuro-Matsuyama et al.; licensee BioMed Central Ltd.

【 图 表 】

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