BioID: A Screen for Protein‐Protein Interactions

互联网2013-12-31

5936

Abstract
Table of Contents
Materials
Figures
Literature Cited

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. 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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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 (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

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; 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 (NH₄ HCO₃ )
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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 19.23.1 Outline of the BioID method. The following steps outline the BioID process. (1 ) Ligate bait cDNA and BioID in‐frame and in an appropriate expression vector. (2 ) Express BioID fusion protein in cells to (3 ) generate stably expressing cells. (4 ) Induce biotinylation with excess biotin prior to (5 ) cell lysis and protein denaturation. (6 ) Perform biotin‐affinity capture of biotinylated proteins. (7 ) Reserve 10% of the sample for streptavidin immunoblot analysis. Use the remainder of the sample for mass spectrometry either by (8A ) SDS‐PAGE separation with (8B ) identification of proteins in isolated bands or (9 ) analysis of peptides released by on‐bead digestion.

View Image

Figure 19.23.2 Analysis of cells stably expressing a BioID‐fusion protein. U2OS cells stably expressing BioID‐lamin A (LaA) were analyzed prior to large‐scale pull‐down experiments. (A ) By fluorescence microscopy it can be observed that all of the cells express similar levels of the myc‐tagged BioID‐LaA protein (red) targeted to the nuclear envelope. Following 24 hr incubation with excess biotin, the vast majority of the biotin signal, detected with streptavidin–Alexa Fluor 488 (green), co‐localizes with the fusion protein. DNA is detected with Hoechst dye (blue). Scale bar is 15 µm. (B ) Following SDS‐PAGE separation, the protein constituencies of BioID‐LaA and control U2OS cells were probed with both streptavidin‐HRP (top) and anti‐myc (bottom). As compared to the few naturally biotinylated proteins in the control sample, there is extensive biotinylation of endogenous proteins in the BioID‐LaA sample.

View Image

Videos

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
	http://www.addgene.org/Kyle_Roux
	BioID plasmids containing epitope‐tagged humanized BirA (R118G) can be obtained from the nonprofit plasmid repository Addgene.
	http://www.sanfordresearch.org/researchcenters/childrenshealth/rouxlab/bioidresources/
	Up‐to‐date information on BioID.