Analysis of Membrane Protein Interactions with a Bacterial Adenylate Cyclase–Based Two‐Hybrid (BACTH) Technique

Scot P. Ouellette1, Gouzel Karimova2, Marilyne Davi2, Daniel Ladant2

1 Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, 2 Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, Paris
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.12
DOI:  10.1002/cpmb.36
Online Posting Date:  April, 2017
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Abstract

The bacterial two‐hybrid (BACTH, for “Bacterial Adenylate Cyclase‐based Two‐Hybrid”) technique is a simple and fast genetic approach to analyze protein‐protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic domain of Bordetella pertussis adenylate cyclase and co‐expressed in strains of Escherichia coli deficient in adenylate cyclase. Association of the hybrid proteins restores synthesis of cyclic AMP (cAMP), which then triggers the expression of catabolic operons such as the lactose operon or the maltose regulon. As BACTH uses a cAMP second messenger, the association between the chimeric proteins can take place at a distance from the transcription machinery. This technique is therefore particularly appropriate for studying interactions involving integral‐membrane or membrane‐associated proteins that may not be soluble in the cytoplasm, and/or that may only associate in the plane of the membrane. This unit describes the basic procedures to characterize protein‐protein interactions with the BACTH genetic system and to search for potential partners of known proteins. © 2017 by John Wiley & Sons, Inc.

Keywords: protein interaction assay; two‐hybrid technique; membrane protein; library screening; Escherichia coli; cAMP signaling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Interaction Between Two Different Proteins with the BACTH Assay
  • Alternate Protocol 1: Gateway Cloning of Genes Encoding Proteins of Interest into BACTHgw Vectors
  • Basic Protocol 2: Application of BACTH for Screening for Interacting Partners of a Target Protein
  • Alternate Protocol 2: Application of BACTHGW in Library Screening
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Interaction Between Two Different Proteins with the BACTH Assay

  Materials
  • BACTH plasmid vectors (see Table 20.12.1; store in TE buffer or distilled H 2O at –20ºC)
  • Competent E. coli cells (e.g., XL1‐Blue from Stratagene)
  • LB agar plates (see recipe)
  • Antibiotics (ampicillin, kanamycin, spectinomycin; see recipe for stock solutions)
  • Plasmid miniprep kit (e.g., Qiagen, Macherey‐Nagel; also see unit 2.4, Wilson, )
  • BACTH reporter strains (see Table 20.12.2; store in LB medium with 10% DMSO at –80ºC); re‐streak on LB‐X‐gal‐IPTG plate (see recipe) before use and start culture from an isolated white colony
  • LB‐X‐gal‐IPTG agar plates (see recipe)
  • LB broth (see recipe)
  • Nalidixic acid (optional)
  • Streptomycin (optional)
  • 0.1 M CaCl 2
  • MacConkey/maltose agar plates (see recipe)
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG; see recipe for stock solution)
  • 5× M63 minimal medium (see recipe)
  • 0.05% (w/v) sodium dodecyl sulfate
  • Chloroform
  • PM2 medium (see recipe)
  • 4 mg/ml o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG), filter sterilized
  • 1 M Na 2CO 3
  • 30° and 37ºC incubator for plates and shaking liquid cultures
  • Spectrophotometer for measuring OD 600
  • 42ºC water bath
  • 96‐well deep‐well blocks (2.2‐ and 1.2‐ml well volumes), sterile
  • Sterile toothpicks
  • AeraSeal sterile adhesive film (Dutscher, cat no. 760215) or Airpore Tape Strips (Qiagen, cat. no. 19571)
  • Rotary shaker (for shaking deep‐well 96‐well blocks)
  • 96‐well flat‐bottom microtiter plates, sterile
  • Microplate reader: Tecan or equivalent plate reader capable of kinetic measurements at wavelengths 405 and 595 (or 600) nm
  • Spreadsheet software (e.g., Microsoft Excel)
  • Additional reagents and equipment for PCR (unit 15.1; Kramer & Coen, ), plasmid miniprep (unit 2.4; Wilson, ), introduction of plasmid DNA into cells (Sambrook, Russell, & Sambrook, ; Seidman, Struhl, Sheen, & Jessen, ), restriction digestion (unit 3.1; Bloch & Grossmann, ), and DNA sequencing (Chapter 7 in this manual)

Alternate Protocol 1: Gateway Cloning of Genes Encoding Proteins of Interest into BACTHgw Vectors

  Additional Materials (also see protocol 1)
  • pDONR221 plasmid (Thermo Fisher Scientific)
  • BP Clonase II enzyme (Thermo Fisher Scientific)
  • Proteinase K
  • BACTH GW destination vectors:
    • pST25‐DEST
    • pSNT25‐DEST
    • pUT18C‐DEST
  • LR Clonase enzyme mix (Thermo Fisher Scientific)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5; Voytas, )

Basic Protocol 2: Application of BACTH for Screening for Interacting Partners of a Target Protein

  Materials
  • BACTH plasmid vectors (see Table 20.12.1)
  • Restriction endonuclease SmaI (also see unit 3.1; Bloch & Grossmann, )
  • Shrimp alkaline phosphatase (Thermo Fisher Scientific, cat. no. 783901000UN)
  • ElectroMAX DH10B cells (Thermo Fisher Scientific; or other highly competent E. coli recipient cells)
  • LB agar plates (see recipe)
  • Antibiotics (ampicillin, kanamycin, spectinomycin; see recipe for stock solutions)
  • LB broth (see recipe)
  • Plasmid miniprep kit (e.g., Qiagen, Macherey‐Nagel; also see unit 2.4, Wilson, )
  • 10% (v/v) glycerol
  • 5× M63 minimal medium (see recipe)
  • M63/maltose plates (see recipe) supplemented with kanamycin (25 μg/ml), ampicillin (50 μg/ml), IPTG (0.5 mM), and X‐Gal (40 μg/ml)
  • Bath sonicator
  • 30° and 37ºC incubator for plates and shaking liquid cultures
  • Spectrophotometer for determining OD 600
  • Refrigerated centrifuge
  • Electroporator (e.g., BioRad) and electroporation cuvettes (1‐mm)
  • 30ºC water bath
  • Additional reagents and equipment for use of mung bean nuclease (unit 3.1; Nichols, ) and DNA polymerases (unit 3.5; Kucera & Nichols, ), restriction digestion (unit 3.1; Bloch & Grossmann, ), cloning into BACTH vectors ( protocol 1), and DNA sequencing (see Chapter 7 in this manual)
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Figures

Videos

Literature Cited

 
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Internet Resources
  http://www.euromedex.com
  Euromedex: Commercial supplier of the BACTH system.
  https://www.thermofisher.com.
  Supplier for Gateway ‘recombineering’ cloning technique.
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