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

BMC Biotechnol. 2013 Mar 28;13:31. doi: 10.1186/1472-6750-13-31.

Abstract

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 degree C 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Escherichia coli / metabolism
  • Fireflies / enzymology*
  • Fluorescence Resonance Energy Transfer
  • Luciferases, Firefly / genetics
  • Luciferases, Firefly / metabolism*
  • Protein Interaction Mapping*
  • Protein Interaction Maps / drug effects
  • Proto-Oncogene Proteins c-mdm2 / chemistry
  • Proto-Oncogene Proteins c-mdm2 / genetics
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Signal-To-Noise Ratio
  • Sirolimus / pharmacology
  • Tacrolimus Binding Protein 1A / chemistry
  • Tacrolimus Binding Protein 1A / genetics
  • Tacrolimus Binding Protein 1A / metabolism
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • Recombinant Fusion Proteins
  • Tumor Suppressor Protein p53
  • Luciferases, Firefly
  • Proto-Oncogene Proteins c-mdm2
  • Tacrolimus Binding Protein 1A
  • Sirolimus