BioID: A Screen for Protein‐Protein Interactions

Kyle J. Roux1, Dae In Kim2, Brian Burke3, Danielle G. May2

1 Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, 2 Children's Health Research Center, Sanford Research, North Sioux Falls, 3 Institute of Medical Biology, Immunos
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.23
DOI:  10.1002/cpps.51
Online Posting Date:  February, 2018
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Abstract

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. © 2018 by John Wiley & Sons, Inc.

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

     
 
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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
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of Cells Expressing a BioID Fusion Protein

  Materials
  • 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
  • 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 (AB7403, Abcam)
  • ABS blocking buffer (see recipe)
  • Enhanced chemiluminescence (ECL) reagent (from commercial source, or see recipe)
  • 30% H 2O 2
  • Antibodies specific to BioID fusion protein (e.g., anti‐myc/HA) or chicken anti‐BioID (BID‐CP‐100, BioFront)
  • 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, Marina, & Yu, ), transfection (Kingston, ), SDS‐PAGE separation (unit 10.1; Gallagher, ), protein transfer (unit 10.7; Goldman, Ursitti, Mozdzanowski, & Speicher, ), and immunoblot detection (unit 10.10; Ni, Xu, & Gallagher, )

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

  Materials
  • 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)
  • Lysis buffer (see recipe)
  • 20% Triton X‐100
  • 50 mM Tris·Cl, pH 7.4 ( appendix 2)
  • Streptavidin Sepharose High Performance Beads (GE Healthcare)
  • Wash buffer (see recipe)
  • 1 mM biotin in 50 mM ammonium bicarbonate (NH 4HCO 3), made freshly
  • 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)
  • Rotator
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Figures

Videos

Literature Cited

  Batsios, P., Meyer, I., & Graf, R. (2016). Proximity‐dependent biotin identification (BioID) in Dictyostelium amoebae. Methods in Enzymology, 569, 23–42. doi: 10.1016/bs.mie.2015.09.007.
  Chan, P., Srikumar, T., Dingar, D., Kalkat, M., Penn, L., & Raught, B. (2014). BioID data of c‐MYC interacting protein partners in culutured cells and xenograft tumors. Data Brief, 1, 76–78. doi: 10.1016/j.dib.2014.10.001.
  Chen, A. L., Kim, E. W., Toh, J. Y., Vashisht, A. A., Rashoff, A. Q., Van, C., … Bradley, P. J. (2015). Novel components of the Toxoplasma inner membrane complex revealed by BioID. MBio, 6(1):e02357‐14. doi: 10.1128/mBio.02357‐14.
  Choi‐Rhee, E., Schulman, H., & Cronan, J. E. (2004). Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Science, 13, 3043–3050. doi: 10.1110/ps.04911804.
  Cronan, J. E. (2005). Targeted and proximity‐dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase. Journal of Nutritional Biochemistry, 16, 416–418. doi: 10.1016/j.jnutbio.2005.03.017.
  De Munter, S., Gornemann, J., Derua, R., Lesage, B., Qian, J., Heroes, E., … Bollen, M. (2017). Split‐BioID: A proximity biotinylation assay for dimerization‐dependent protein interactions. FEBS Letters, 591(2)415–424. doi: 10.1002/1873‐3468.12548.
  Donaldson, J. G. (2001). Immunofluorescence staining. Current Protocols in Cell Biology, 00, 4.3.1–4.3.6.
  Elion, E. A., Marina, P., & Yu, L. (2007). Constructing recombinant DNA molecules by PCR. Current Protocols in Molecular Biology, 78, 3.17.1–3.17.12.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1:10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Goldman, A., Ursitti, J. A., Mozdzanowski, J., & Speicher, D.W. (2015). Electroblotting from polyacrylamide gels. Current Protocols in Protein Science, 82, 10.7.1‐10.7.16. doi: 10.1002/0471140864.ps1007s82.
  Kim, D. I., Birendra, K. C., Zhu, W., Motamedchaboki, K., Doye, V., & Roux, K. J. (2014). Probing nuclear pore complex architecture with proximity‐dependent biotinylation. Proceedings of the National Academy of Sciences of the United States of America, 111(24), E2453–2461. doi: 10.1073/pnas.1406459111.
  Kim, D. I., Jensen, S. C., Noble, K. A., Birendra, K. C., Roux, K. H., Motamedchaboki, K., & Roux, K. J. (2016). An improved smaller biotin ligase for BioID proximity labeling. Molecular Biology of the Cell, 27(8), 1188–1196. doi: 10.1091/mbc.E15‐12‐0844.
  Kim, D. I., & Roux, K. J. (2016). Filling the void: Proximity‐based labeling of proteins in living cells. Trends in Cell Biology, 26(11), 804–817. doi: 10.1016/j.tcb.2016.09.004.
  Kingston, R. E. (2003). Introduction of DNA into mammalian cells. Current Protocols in Molecular Biology, 9.0.1–9.0.5.
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  Ni, D., Xu, P., and Gallagher, S. (2017). Immunoblotting and immunodetection. Current Protocols in Protein Science, 88, 10.10.1‐10.10.37. doi: 10.1002/cpps.32.
  Opitz, N., Schmitt, K., Hofer‐Pretz, V., Neumann, B., Krebber, H., Braus, G. H., … Valerius, O. (2017). Capturing the Asc1p/RACK1 microenvironment at the head region of the 40S ribosome with quantitative BioID in yeast. Molecular and Cell Proteomics, pii: mcp.M116.066654. doi: 10.1074/mcp.M116.066654.
  Roux, K. J., Kim, D. I., Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. Journal of Cell Biology, 196, 801–810. doi: 10.1083/jcb.201112098.
  Uezu, A., Kanak, D. J., Bradshaw, T. W., Soderblom, E. J., Catavero, C. M., Burette, A. C., … Soderling, S. H. (2016). Identification of an elaborate complex mediating postsynaptic inhibition. Science, 353(6304), 1123–1129. doi: 10.1126/science.aag0821.
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Internet Resources
  https://www.addgene.org/Kyle_Roux
  BioID and BioID2 plasmids can be obtained from the non‐profit plasmid repository Addgene.
  https://www.sanfordresearch.org/researchcenters/childrenshealth/rouxlab/bioidresources/
  Up‐to‐date information on BioID.
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