Quantitative Analysis of Protein‐Protein Interactions by Split Firefly Luciferase Complementation in Plant Protoplasts

Jian‐Feng Li1, Dandan Zhang1

1 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.9
DOI:  10.1002/0471142727.mb2009s107
Online Posting Date:  July, 2014
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This unit describes the split firefly luciferase complementation (SFLC) assay, a high‐throughput quantitative method that can be used to investigate protein‐protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell‐permeable substrate D‐luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real‐time manner. Another advantage is the unusually high DNA co‐transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method. Curr. Protoc. Mol. Biol. 107:20.9.1‐20.9.8. © 2014 by John Wiley & Sons, Inc.

Keywords: protein‐protein interaction; plant protoplasts; split firefly luciferase complementation

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Table of Contents

  • Introduction
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • Arabidopsis plants: 4‐5 weeks old, Arabidopsis ecotype Col‐0 (seeds available from Arabidopsis Biological Resource Center) grown on Jiffy7 soil (Jiffy Group, http://www.jiffygroup.com) under conditions of 65% humidity and 75 μmol m−2 sec−1 light intensity in photoperiods of 12 hr light at 23°C and 12 hr dark at 20°C
  • Cell wall digestion solution (see recipe)
  • W5 solution (see recipe)
  • MMg solution (see recipe)
  • 2 μg/μl DNA purified from:
    • Individual split luciferase fusion expression plasmids (protein A−FLucN and protein B−FLucC; Fig. A)
    • Internal control UBQ:GUS plasmid
    • Positive and negative control plasmids (see Critical Parameters and Troubleshooting)
  • PEG solution (see recipe)
  • 5% (v/v) bovine calf serum (HyClone, cat. no. SH30072.03)
  • WI solution (see recipe)
  • 2.5 mg/ml D‐luciferin (Research Products International, cat. no. L37060; stock solution can be stored up to 1 year at −80°C)
  • Protoplast lysis solution (see recipe)
  • MUG solution (see recipe)
  • Razor blades (VWR Scientific, cat. no. 55411‐055)
  • Clean white paper (e.g., printer paper)
  • Sterile disposable inoculating loop (BD Biosciences, cat. no. 220217)
  • 100 × 25−mm2 Petri dish (VWR Scientific, cat. no. 25389‐000)
  • Horizontal shaker (VWR, cat. no. 89032‐092)
  • Nylon mesh (Carolina Biological Supplies, cat. no. 652222N)
  • 30‐ml round‐bottom tube (Sarstedt, cat. no. 55.517)
  • CL2 clinical centrifuge (Thermo Scientific, cat. no. 004260F)
  • Hemacytometer (Hausser Scientific, cat. no. 1483)
  • 2‐ml round‐bottom microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • 24‐well culture plate (Fisher Scientific, cat. no. 08‐772‐1)
  • 96‐well plates with black frame and white wells (PerkinElmer, cat. no. 6005039)
  • 12‐channel micropipettors (Thermo Scientific, cat. no. 4662070 and 4662040)
  • GloMax‐Multi multimode microplate reader (Promega, cat. no. E7041)
  • Microseal B adhesive seal (Bio‐Rad, cat. no. MSB‐1001)
  • Digital vortex mixer with microplate holder (VWR, cat. no. TY‐LP‐945311 and 12620‐878)
  • Benchtop microplate centrifuge (Beckman, cat. no. B06314)
NOTE: All steps are conducted at room temperature unless otherwise stated.
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Literature Cited

Literature Cited
  Arabidopsis Interactome Mapping Consortium. 2011. Evidence for network evolution in an Arabidopsis interactome map. Science 333:601‐607.
  Chen, H., Zou, Y., Shang, Y., Lin, H., Wang, Y., Cai, R., Tang, X., and Zhou, J.M. 2008. Firefly luciferase complementation imaging assay for protein‐protein interactions in plants. Plant Physiol. 146:368‐376.
  Chen, J., Lalonde, S., Obrdlik, P., Noorani Vatani, A., Parsa, S.A., Vilarino, C., Revuelta, J.L., Frommer, W.B., and Rhee, S.Y. 2012. Uncovering Arabidopsis membrane protein interactome enriched in transporters using mating‐based split ubiquitin assays and classification models. Front. Plant Sci. 3:124.
  Gehl, C., Kaufholdt, D., Hamisch, D., Bikker, R., Kudla, J., Mendel, R.R., and Hänsch, R. 2011. Quantitative analysis of dynamic protein‐protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors. Plant J. 67:542‐553.
  Ladonde, S., Ehrhardt, D.W., Loque, D., Chen, J., Rhee, S.Y., and Frommer, W.B. 2008. Molecular and cellular approaches for the detection of protein‐protein interactions: Latest techniques and current limitations. Plant J. 53:610‐635.
  Lee, L.Y., Wu, F.H., Hsu, C.T., Shen, S.C., Yeh, H.Y., Liao, D.C., Fang, M.J., Liu, N.T., Yen, Y.C., Dokladal, L., Sykorova, E., Gelvin, S.B., and Lin, C.S. 2012. Screening a cDNA library for protein‐protein interactions directly in planta. Plant Cell 24:1746‐1759.
  Li, J.F., Bush, J., Xiong, Y., Li, L., and McCormack, M. 2011. Large‐scale protein‐protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation. PLoS One 6:e27364.
  Luker, K.E., Smith, M.C.P., Luker, G.D., Gammon, S.T., Piwnica‐Worms, H., and Piwnica‐Worms, D. 2004. Kinetics of regulated protein‐protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. U.S.A. 101:12288‐12293.
  Müller, S.M., Galliardt, H., Schneider, J, Barisas, B.G., and Seidel, T. 2013. Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Front. Plant Sci. 4:413
  Paulmurugan, R. and Gambhir, S.S. 2007. Combinatorial library screening for developing an improved split‐firefly luciferase fragment‐assisted complementation system for studying protein‐protein interactions. Anal. Chem. 79:2346‐2353.
  Porter, J.R., Stains, C.I., Jester, B.W., and Ghosh, I. 2008. A general and rapid cell‐free approach for the interrogation of protein‐protein, protein‐DNA, and protein‐RNA interactions and their antagonists utilizing split‐protein reporters. J. Am. Chem. Soc. 130:6488‐6497.
  Yoo, S.D., Cho, Y.H., and Sheen, J. 2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2:1565‐1572.
Key Reference
  Li et al., 2011. See above.
  Demonstrates the ease and power of the SFLC assay for large‐scale protein‐protein interaction analyses in Arabidopsis mesophyll protoplasts.
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