Recombinant Protein Complex Expression in E. coli

William Selleck1, Song Tan1

1 Center for Gene Regulation, Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.21
DOI:  10.1002/0471140864.ps0521s52
Online Posting Date:  May, 2008
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Abstract

This unit provides procedures to design, create, and utilize polycistronic plasmids that express multicomponent protein complexes in E. coli. Both the original pST39 polycistronic expression system, which permits four genes to be coexpressed from a single plasmid, and the more recent pST44 polycistronic system, which facilitates incorporation of affinity tags and simplifies the construction of variant deletion or point mutation polycistronic plasmids, are described. Emphasis is placed on practical details for creating polycistronic expression plasmids, expressing the protein complex in E. coli, purifying the protein complex, and troubleshooting potential expression problems. Curr. Protoc. Protein Sci. 52:5.21.1‐5.21.21. © 2008 by John Wiley & Sons, Inc.

Keywords: multisubunit protein complexes; polycistronic expression; affinity tags

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Small‐Scale Expression of Subunits and Complex in E. coli
  • Basic Protocol 2: Small‐Scale Purification of Tagged Subunits and Complex
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Small‐Scale Expression of Subunits and Complex in E. coli

  Materials
  • Expression plasmid (see )
  • Competent E. coli host strain such as BL21(DE3)pLysS
  • 2×TY medium (see recipe) without antibiotics
  • TYE plates (see recipe) containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol
  • 2×TY medium (see recipe) with 50 µg/ml ampicillin and 25 µg/ml chloramphenicol
  • 1× SDS sample buffer (see recipe)
  • 0.2 M isopropyl‐β‐thiogalactopyranoside (IPTG) in H 2O (filter through 0.22‐µm filter and store up to 1 year at –20°C)
  • Appropriate buffer for downstream purification steps: e.g., P300‐EDTA buffer (see recipe) or T100 buffer (see recipe)
  • Liquid nitrogen
  • 42°C water bath
  • Shaking incubator (e.g., New Brunswick model C24)
  • Spectrophotometer to measure optical density at 600 nm
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Tabletop centrifuge
  • Additional reagents and equipment for preparing E. coli cells for analysis by SDS‐PAGE (unit 5.2, Support Protocol 4) and SDS‐PAGE (unit 10.1)

Basic Protocol 2: Small‐Scale Purification of Tagged Subunits and Complex

  Materials
  • Affinity chromatography resin: e.g., Talon metal affinity resin (Clontech)
  • P300‐EDTA buffer (see recipe)
  • Soluble cellular extract (see protocol 1, step 6)
  • P300‐EDTA buffer containing 100 mM imidazole (see recipe)
  • 15‐ml conical polypropylene centrifuge tubes (Falcon)
  • Tabletop centrifuge
  • Sonicator: Any sonicator capable of disrupting E. coli cells will suffice, e.g., Branson S‐450D 400‐W probe sonicator with 1/2‐in. probe)
  • Bio‐Spin disposable spin columns (BioRad catalog #732‐6008)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), staining of proteins in gels (unit 10.5), and immunoblotting (unit 10.10)
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Figures

Videos

Literature Cited

Literature Cited
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