Bioluminescence Resonance Energy Transfer (BRET)‐Based Synthetic Sensor Platform for Drug Discovery

Jongchan Woo1, Jason Hong1, Savithramma P. Dinesh‐Kumar1

1 Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.30
DOI:  10.1002/cpps.30
Online Posting Date:  April, 2017
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Abstract

Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein‐protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro. Here, we report the methods for constructing a biosensor of human HsLC3a as a probe for autophagy biogenesis and the optimization of the intramolecular BRET assay that allows for high‐throughput screening of chemical modulators of autophagy. User‐friendly working interface with the BRET‐based synthetic sensor of HsLC3a makes drug discovery easy and amenable for high‐throughput. The BRET protocol described here could be easily applicable to generate other biosensors for monitoring PPIs by measurement of intermolecular BRET. © 2017 by John Wiley & Sons, Inc.

Keywords: autophagy; bioluminescence resonance energy transfer (BRET); drug discovery; HsLC3a; HsATG4; and luciferase; protein‐protein interactions (PPIs)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chemical Library Screening for HsATG4b‐Mediated Processing of HsLC3a‐Sensor
  • Alternate Protocol 1: In Vitro Cleavage Assay by SDS‐PAGE and Immunoblotting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Chemical Library Screening for HsATG4b‐Mediated Processing of HsLC3a‐Sensor

  Materials
  • Purified HsATG4b and HsLC3a‐sensor proteins (Seo et al., ; Woo et al., )
  • Ice
  • Coelenterazine (CLZ; see recipe)
  • Phosphate‐buffered saline (PBS) solution, pH 7.4 (e.g., VWR Life Science, cat no. E504‐500 ML)
  • Dimethyl sulfoxide (Sigma‐Aldrich, cat no. 472301‐500 ML)
  • Small‐molecule chemical library (user defined)
  • PCR 8‐tube strips (e.g., USA Scientific, cat no. 1402‐2700)
  • PCR tube rack
  • Aluminum foil
  • Multichannel pipette capable of handling 1 to 100 µl volumes
  • Multichannel pipette reservoir trough (e.g., VWR, cat no. 89094‐662)
  • 96‐well white, flat bottom, tissue culture treated, polystyrene plates (e.g., Corning, cat no. 3917)
  • Adhesive aluminum sealing film for 96‐well plates (e.g., ThermoFisher Scientific, cat no. AB0626)
  • Centrifuge
  • Plate reader (e.g., TECAN Infinite 200)
  • Excel software (Microsoft Office, Microsoft)

Alternate Protocol 1: In Vitro Cleavage Assay by SDS‐PAGE and Immunoblotting

  Materials
  • Purified HsATG4b and HsLC3a‐sensor proteins (Seo et al., ; Woo et al., )
  • Dimethyl sulfoxide (DMSO; e.g., Sigma‐Aldrich, cat no. 472301‐500 ML)
  • Small‐molecule chemical library (user defined)
  • 2× Laemmli sample buffer (e.g., Bio‐rad cat no. 1610737)
  • SDS‐PAGE gel (e.g., Mini‐Protean TGX Precast Gels 10%; Bio‐rad, cat no. 4561034)
  • Protein marker (e.g., Spectra Multicolor Broad Range Protein Ladder, cat no. 26634)
  • 10× Tris/Glycine/SDS Buffer (see recipe)
  • Cathode buffer (see recipe)
  • Anode I and II buffers (see reciperecipes)
  • Methanol (e.g., Fisher Chemical, cat no. A412Sk‐4)
  • Distilled water
  • 5% and 3% nonfat milk (see recipes)
  • Primary antibody anti‐Renilla luciferase, clone 5B11.2 (e.g., EMD Millipore, cat no. MAB4400)
  • Phosphate‐buffered saline (PBS) solution, pH 7.4 (e.g., VWR Life Science, cat no. E504‐500 ML)
  • Tween‐20 (e.g., VWR Life Science, cat no. 0777‐1 liter)
  • Secondary antibody, anti‐Mouse IgG (whole molecule)‐peroxidase antibody produced in rabbit (e.g., Sigma‐Aldrich, cat no. A9044‐2 ML)
  • SuperSignal West Pico Chemiluminescent Substrate (e.g., ThermoFisher Scientific, cat no. 34077)
  • 50‐ml conical tubes (e.g., Corning, cat no. 430290)
  • Centrifuge
  • Water bath
  • Floating tube rack
  • Electrophoresis chamber (e.g., Mini‐PREOTEAN Tetra Vertical Electrophoresis Cell; cat no. 1658004)
  • Electrophoresis power supply (e.g., Bio‐Rad, PowerPac HC High‐Current Power Supply)
  • Orbital shaker (e.g., VWR Orbital Shaker, cat no. DS‐500E)
  • Plates (any plastic 4 in × 4 in container that can hold membrane and liquid solutions)
  • PVDF transfer membranes (e.g., Immobilon, cat no. IPVH00010)
  • Plate tray
  • Thick blot filter paper (e.g., Bio‐Rad, cat no. 1650921)
  • Transfer system (e.g., Trans‐Blot Turbo Transfer System; Bio‐Rad, cat no. 1704155)
  • Tweezers
  • Paper towels
  • Sheet protector non‐glare simple loading 8.5 in × 11 in (e.g., Office Depot, cat no. 535038)
  • Beakers (small and large)
  • 1.5‐ml microcentrifuge tubes
  • X‐ray film (e.g., Research Products International, cat no. 248300)
  • X‐ray film cassette
  • Film developer machine with dark room for film developing
  • Stir bars
  • Hot plate

Support Protocol 1:

  Materials
  • Film (see protocol 2Alternate Protocol)
  • Image scanner
  • ImageJ program (e.g., Fiji program, NIH)
  • Excel program
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Figures

Videos

Literature Cited

Literature Cited
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