BioID: A Screen for Protein‐Protein Interactions

Kyle J. Roux1, Dae In Kim2, Brian Burke3

1 University of South Dakota, Sioux Falls, South Dakota, 2 Children's Health Research Center, Sanford Research, North Sioux Falls, South Dakota, 3 Institute of Medical Biology, Immunos
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.23
DOI:  10.1002/0471140864.ps1923s74
Online Posting Date:  November, 2013
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BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin‐affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. Curr. Protoc. Protein Sci. 74:19.23.1‐19.23.14. © 2013 by John Wiley & Sons, Inc.

Keywords: BioID; proximity‐dependent labeling; protein‐protein interaction; biotinylation

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of Cells Expressing a BioID Fusion Protein
  • Basic Protocol 2: BioID Pull‐Down to Identify Candidate Proteins
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Generation of Cells Expressing a BioID Fusion Protein

  • Expression plasmid for BioID fusion protein (see Internet Resources)
  • Gene for protein of interest
  • Cells of choice for transfection and appropriate medium
  • 1 mM (20×) biotin (see recipe)
  • Fixative, e.g., paraformaldehyde (PFA)
  • Triton X‐100 (TX‐100)
  • Streptavidin‐Alexa Fluor (Invitrogen)
  • DNA labeling reagent (e.g., Hoechst, DAPI)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • SDS‐PAGE sample buffer (see recipe)
  • BSA blocking buffer (see recipe)
  • Streptavidin‐HRP (High Sensitivity Streptavidin‐HRP, Thermo Scientific)
  • ABS blocking buffer (see recipe)
  • Enhanced chemiluminescence (ECL) reagent (from commercial source, or see recipe)
  • Quenching solution (see recipe)
  • Antibodies specific to BioID fusion protein (e.g., anti‐myc/HA) or chicken anti‐BirA ab14002 (Abcam)
  • Secondary antibodies to detect chosen primary antibody (Alexa Fluor form and HRP‐conjugated form)
  • 6‐well plates
  • Glass coverslips
  • Sonicator
  • SDS‐PAGE electrophoresis unit (Mini‐PROTEAN II Electrophoresis Cell, Bio‐Rad)
  • Semi‐dry transfer cell (Trans‐Blot, Bio‐Rad)
  • Additional reagents and equipment for PCR cloning (Elion et al., ), transfection (Kingston, ), SDS‐PAGE separation (unit 10.1), protein transfer (unit 10.7), and immunoblot detection (unit 10.10)

Basic Protocol 2: BioID Pull‐Down to Identify Candidate Proteins

  • Four 10‐cm dishes of cells for each experimental condition (cells expressing BioID constructs or control cells)
  • Complete medium
  • 1 mM (20×) biotin (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Lysis buffer (see recipe)
  • 20% Triton X‐100
  • 50 mM Tris·Cl, pH 7.4 ( appendix 2E)
  • Dynabeads (MyOne Streptavidin C1, Life Technologies)
  • Wash buffer 1 (see recipe)
  • Wash buffer 2 (see recipe)
  • Wash buffer 3 (see recipe)
  • 50 mM ammonium bicarbonate (NH 4HCO 3)
  • 1× SDS‐PAGE sample buffer (see recipe)
  • DNase/RNase‐free tubes, 15 ml conical and 2 ml microcentrifuge
  • Tube cap opener
  • Sonicator (Branson Sonifier‐250 or equivalent)
  • MagneSphere Technology Magnetic Separation Stand (Promega)
  • Rotator
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Literature Cited

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.
  Comartin, D., Gupta, G.D., Fussner, E., Coyaud, E., Hasegan, M., Archinti, M., Cheung, S.W., Pinchev, D., Lawo, S., Raught, B., Bazett‐Jones, D.P., Luders, J., and Pelletier, L. 2013. CEP120 and SPICE1 cooperate with CPAP in centriole elongation. Curr. Biol. 23:1360‐1366.
  Cronan, J.E. 2005. Targeted and proximity‐dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase. J. Nutr. Biochem. 16:416‐418.
  Donaldson, J.G. 2001. Immunofluorescence staining. Curr. Protoc. Cell Biol. 00:4.3.1‐4.3.6.
  Elion, E.A., Marina, P., and Yu, L. 2007. Constructing recombinant DNA molecules by PCR. Curr. Protoc. Mol. Biol. 78:3.17.1‐3.17.12.
  Kingston, R.E. 2003. Introduction of DNA into mammalian cells. Curr. Protoc. Mol. Biol. 9.0.1‐9.0.5.
  Kwon, K. and Beckett, D. 2000. Function of a conserved sequence motif in biotin holoenzyme synthetases. Protein Sci. 9:1530‐1539.
  Morriswood, B., Havlicek, K., Demmel, L., Yavuz, S., Sealey‐Cardona, M., Vidilaseris, K., Anrather, D., Kostan, J., Djinovic‐Carugo, K., Roux, K.J., and Warren, G. 2013. Novel bilobe components in Trypanosoma brucei identified using proximity‐dependent biotinylation. Eukaryot. Cell 12:356‐367.
  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.
  Van Itallie, C.M., Aponte, A., Tietgens, A.J., Gucek, M., Fredriksson, K., and Anderson, J.M. 2013. The N and C termini of ZO‐1 are surrounded by distinct proteins and functional protein networks. J. Biol. Chem. 288:13775‐13788.
  van Steensel, B. and Henikoff, S. 2000. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18:424‐428.
Internet Resources
  BioID plasmids containing epitope‐tagged humanized BirA (R118G) can be obtained from the nonprofit plasmid repository Addgene.
  Up‐to‐date information on BioID.
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