Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC)

Lisette G.G.C. Verhoef1, Mark Wade1

1 Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Milan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 30.5
DOI:  10.1002/cpps.42
Online Posting Date:  November, 2017
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Abstract

The number of intracellular protein‐protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed ‘bimolecular complementation’ relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins. When these proteins interact, they effectively reconstitute the activity of the molecular reporter (typically leading to increased fluorescence or luminescence). This unit describes the selection and development of bimolecular luminescence complementation (BiLC) assays for reporting intracellular PPIs, and provides examples in which BiLC was used to identify small molecules that can modulate PPIs. © 2017 by John Wiley & Sons, Inc.

Keywords: BiLC; protein‐protein interaction; luciferase; cellular assay; screen

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of BiLC Constructs and Validation of BiLC Signals
  • Basic Protocol 2: Generation of BiLC Stable Cell Lines
  • Basic Protocol 3: Combined Use of Firefly and NanoLuc to Measure Multiple Protein‐Protein Interactions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of BiLC Constructs and Validation of BiLC Signals

  Materials
  • BiLC vector DNA: For transient transfection studies to test BiLC partner pairs, an expression vector such as pCDNA3.1 is suitable. Pairs that produce the greatest BiLC signal can then be subcloned to other vectors, such as a doxycycline‐inducible system (see protocol 2 and Li et al., ).
  • Transfectable cells: These are cells that can be transfected with high efficiency, such as HEK293 and U2OS, and that are suitable for testing BiLC pairs. Note that if the BiLC assays will be used in protocols that require several exchanges of medium, then cell lines that adhere well—U2OS or SaOS2—should be used in preference to HEK293, which are loosely adherent.
  • Lipid‐based transfection reagent (e.g., LipoFectamine2000, ThermoFisher)
  • Dulbecco's phosphate‐buffered saline (DPBS) with Ca2+ and Mg2+ (e.g., Gibco, ThermoFisher)
  • RIPA (radioimmunoprecipitation) lysis buffer (see recipe)
  • Luciferase detection reagent (SteadyGlo or NanoGlo from Promega)
  • 6‐well plates (Corning Costar, cat. no. 3506, or similar)
  • 4 × 10–cm tissue culture plates
  • Assay plates: 96‐well solid white (Corning, cat. no. 3917) and 96‐well black clear‐bottom (Corning, cat. no. 3603)
  • Refrigerated centrifuge
  • Luminometer: Many commercial luminometers can be used to detect the signal from BiLC. These include Promega's GloMax, TECAN's Safire2 (now TECAN M1000), BMG's Pherastar, BioTek's Synergy series, Perkin Elmer Envision.
  • Additional reagents and equipment for transfection of cells (unit 5.10; Chen, Gray, Ma, & Subramanian, ), cell culture including trypsinization and counting cells ( appendix 3C; Phelan, ), and western blotting (unit 10.10; Ni, Xu, and Gallagher, )

Basic Protocol 2: Generation of BiLC Stable Cell Lines

  Materials
  • Cell line used for optimization of the BiLC pair ( protocol 1)
  • G418 and puromycin selection antibiotics
  • Cell growth medium
  • Dulbecco's phosphate‐buffered saline (DPBS) with Ca2+ and Mg2+ (e.g., Gibco, ThermoFisher)
  • Smooth silicone grease
  • 0.25% trypsin/EDTA (e.g., Gibco)
  • Dox‐responsive plasmid (e.g., pTRE3G‐luc)
  • 500 ng/ml doxycycline
  • pCMV‐Tet3G (Clontech, cat. no. 631168)
  • Linearized vector that contains a constitutively expressed puromycin resistance gene
  • 6‐well plates (Corning Costar, cat. no. 3506, or similar)
  • 4 × 10–cm tissue culture plates (e.g., Corning Costar)
  • 24‐well plates (e.g., Corning Costar)
  • Tissue culture microscope
  • Cloning cylinders (Sigma, cat. no. C1059)
  • Sterile forceps
  • Additional reagents and equipment for transfection of cells, including use of selection agent (unit 5.10; Chen et al., ), cell culture including trypsinization and counting cells ( appendix 3C; Phelan, ), and detection of luciferase activity ( protocol 1, steps 5 and 6)

Basic Protocol 3: Combined Use of Firefly and NanoLuc to Measure Multiple Protein‐Protein Interactions

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Cells of interest
  • NanoGlo Dual Luciferase Reporter Assay (Promega) or NanoGlo detection reagent (Promega)
  • Assay plates: 384‐well solid white (Corning, cat. no. 3570)
  • Multiplex Firefly and NanoLuc in a 384‐well format
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Figures

Videos

Literature Cited

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