Protein-Protein Interaction: Tandem Affinity Purification in Bacteria

Methods Mol Biol. 2017;1615:221-232. doi: 10.1007/978-1-4939-7033-9_18.

Abstract

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.

Keywords: Affinity purification; Calmodulin binding peptide (CBP); Escherichia coli; ProtA; Protein complex; Protein–protein interaction; Salmonella; Tandem affinity purification (TAP); Tobacco etch virus (TEV).

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Western
  • Chromatography, Affinity
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression
  • Gene Order
  • Genetic Vectors / genetics
  • Immunoprecipitation
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / isolation & purification
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Proteins / chemistry
  • Proteins / isolation & purification*
  • Proteins / metabolism
  • Proteomics
  • Recombinant Fusion Proteins
  • Tandem Mass Spectrometry

Substances

  • Multiprotein Complexes
  • Proteins
  • Recombinant Fusion Proteins