Proximity‐Dependent Biotinylation for Identification of Interacting Proteins

Valerie Le Sage1, Alessandro Cinti2, Andrew J. Mouland2

1 HIV‐1 RNA Trafficking Laboratory, Lady Davis Institute at the Jewish General Hospital, Montréal, Québec, 2 Department of Medicine, McGill University, Montréal, Québec
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.19
DOI:  10.1002/cpcb.11
Online Posting Date:  December, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity‐dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein‐protein and proximity‐based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins. In vivo protein‐protein interactions can be very transient and occur momentarily to facilitate signaling or a metabolic function. BioID is useful in identifying these weak and/or transient interactions that are not detected by traditional methods such as yeast two‐hybrid or affinity purification. Here, we outline a BioID protocol that can be used as a workflow to guide a new application. © 2016 by John Wiley & Sons, Inc.

Keywords: BioID; BirA; proximity biotinylation; protein‐protein interactions

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Design and Creation of a BioID Fusion Protein and Validation of Protein Expression
  • Basic Protocol 2: Biotinylation and Identification of Bait‐Interacting Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Design and Creation of a BioID Fusion Protein and Validation of Protein Expression

  • Gene for protein of interest
  • Expression plasmids for BioID or BioID2 fusion protein (Addgene #36047, #35700, #74223, or #74224)
  • Appropriate cell type and culture medium
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% (w/v) paraformaldehyde in PBS
  • 0.1 M glycine in PBS
  • 0.2% (v/v) Triton X‐100 in PBS
  • Primary antibodies against the bait protein of interest (anti‐myc/HA antibodies or chicken anti‐BirA; e.g., Abcam, cat. no. ab14002)
  • Alexa Fluor‐conjugated secondary antibody
  • Alexa Fluor‐conjugated streptavidin
  • Blocking solution for immunofluorescence (see recipe)
  • DNA labeling reagent for immunofluorescence (DAPI or Hoechst)
  • RIPA lysis buffer (see recipe)
  • 1× SDS‐PAGE sample buffer (see recipe)
  • Nitrocellulose or PVDF membrane
  • Tris‐buffered saline containing Tween 20 (TBST; see recipe)
  • 5% (w/v) non‐fat milk blocking buffer (see recipe)
  • 0.5% (w/v) bovine serum albumin (BSA) in TBST
  • Streptavidin‐HRP antibody
  • Enhanced chemiluminescence (ECL) reagent
  • 12‐well and 6‐well plates
  • Glass coverslips and slides
  • 95°C heating block
  • Additional reagents and equipment for gene cloning ( appendix 3F), determination of protein concentration ( appendix 3H), SDS‐PAGE electrophoresis (unit 6.1), and immunoblotting (unit 6.2)

Basic Protocol 2: Biotinylation and Identification of Bait‐Interacting Proteins

  • Cells stably expressing the BioID‐bait fusion protein and control cells expressing the BioID tag alone
  • Appropriate cell culture medium for cell line
  • Complete medium (cell culture medium supplemented with 50 µM biotin; see recipe)
  • PBS (see recipe)
  • RIPA lysis buffer (see recipe)
  • Protease inhibitors (e.g., Roche cOmplete)
  • Wash buffer 1 (see recipe)
  • Wash buffer 2 (see recipe)
  • Wash buffer 3 (see recipe)
  • 50 mM Tris⋅Cl, pH 7.4 (see appendix 2A)
  • 1× SDS‐PAGE sample buffer (see recipe)
  • 10‐cm dishes
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Dynabeads (e.g., Life Technologies, MyOne Streptavidin C1)
  • Magnet/magnetic rack
  • Mixing device (tilting or rotation)
  • 95°C heating block
  • Additional reagents and equipment for determination of protein concentration by Bradford assay ( appendix 3H) and gel electrophoresis (unit 6.1)
PDF or HTML at Wiley Online Library



Literature Cited

  Choi‐Rhee, E., Schulman, H., and Cronan, J.E. 2004. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13:3043‐3050. doi: 10.1110/ps.04911804.
  Gallagher, S.R. 2007. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Cell Biol. 37:6.1.1‐6.1.38. doi: 10.1002/0471143030.cb0601s37.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J. G. 2011. Immunoblotting and immunodetection. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28. doi: 10.1002/0471143030.cb0602s52.
  Kim, D.I., Birendra, K.C., Zhu, W., Motamedchaboki, K., Doye, V., and Roux, K.J. 2014. Probing nuclear pore complex architecture with proximity‐dependent biotinylation. Proc. Natl. Acad. Sci. U.S.A. 111:E2453‐2461. doi: 10.1073/pnas.1406459111.
  Kim, D.I., Jensen, S.C., Noble, K.A., Kc, B., Roux, K.H., Motamedchaboki, K., and Roux, K.J. 2016. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27:1188‐1196. doi: 10.1091/mbc.E15‐12‐0844.
  Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Cell Biol. 10:A.3F.1‐A.3F.14. doi: 10.1002/0471143030.cba03fs10.
  Krohn, R.I. 2011. The colorimetric detection and quantitation of total protein. Curr. Protoc. Cell Biol. 52:A.3H.1‐A.3H.28. doi: 10.1002/0471143030.cba03hs52.
  Kwon, K. and Beckett, D. 2000. Function of a conserved sequence motif in biotin holoenzyme synthetases. Protein Sci. 9:1530‐1539. doi: 10.1110/ps.9.8.1530.
  Lambert, J.P., Tucholska, M., Go, C., Knight, J.D., and Gingras, A.C. 2015. Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin‐associated protein complexes. J. Proteomics 118:81‐94. doi: 10.1016/j.jprot.2014.09.011.
  Le Sage, V., Cinti, A., Valiente‐Echeverria, F., and Mouland, A.J. 2015. Proteomic analysis of HIV‐1 Gag interacting partners using proximity‐dependent biotinylation. Virol. J. 12:138. doi: 10.1186/s12985‐015‐0365‐6.
  Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and Ting, A.Y. 2013. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339:1328‐1331. doi: 10.1126/science.1230593.
  Ritchie, C., Cylinder, I., Platt, E.J., and Barklis, E. 2015. Analysis of HIV‐1 Gag protein interactions via biotin ligase tagging. J. Virol. 89:3988‐4001. doi: 10.1128/JVI.03584‐14.
  Roux, K.J., Kim, D.I., Raida, M., and Burke, B. 2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196:801‐810. doi: 10.1083/jcb.201112098.
  Tron, C.M., McNae, I.W., Nutley, M., Clarke, D.J., Cooper, A., Walkinshaw, M.D., Baxter, R.L., and Campopiano, D.J. 2009. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity. J. Mol. Biol. 387:129‐146. doi: 10.1016/j.jmb.2008.12.086.
  Wilson, K.P., Shewchuk, L.M., Brennan, R.G., Otsuka, A.J., and Matthews, B.W. 1992. Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin‐ and DNA‐binding domains. Proc. Natl. Acad. Sci. U.S.A. 89:9257‐9261. doi: 10.1073/pnas.89.19.9257.
Internet Resources
  Addgene plasmid repository for the BioID and BioID2 plasmids.
PDF or HTML at Wiley Online Library