Selection of Escherichia coli Expression Systems

Alain Bernard1, Mark Payton1

1 Glaxo Institute for Molecular Biology, Geneva
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.2
DOI:  10.1002/0471140864.ps0502s00
Online Posting Date:  May, 2001
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Abstract

This unit lists the most useful expression strains of E. coli for fermentation processes. Standard procedures are provided for several expression systems, namely, temperature induction via the pL promoter and chemical induction via the trp promoter, lac or tac promoters, and the T7 promoter. These protocols require that the gene encoding the protein of interest has been identified and cloned into an appropriate expression vector using standard molecular biology techniques. Transformation of a suitable host strain (e.g., by electroporation) is also described and is a prerequisite. Protocols for the analysis of plasmid stability and subsequent storage are provided. Support protocols describe how to prepare samples for electrophoresis, how to analyze the solubility of the expressed proteins, and how to make samples of periplasmic extracts and extracellular media (using TCA precipitation). Many of the support protocols are small‐scale analysis procedures that are used to guide subsequent purification strategies and determine the suitability of the expression system for further development and scale‐up.

     
 
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Table of Contents

  • Basic Protocol 1: Expression Under Control of pL Promoter: Temperature Induction
  • Basic Protocol 2: Expression Under Control of trp Promoter: Chemical Induction
  • Alternate Protocol 1: Expression Under Control of lac/tac Promoters
  • Alternate Protocol 2: T7 RNA Polymerase/Promoter Expression System
  • Support Protocol 1: Transformation by Electroporation
  • Support Protocol 2: Plasmid Stability Analysis
  • Support Protocol 3: Strain Storage
  • Support Protocol 4: Preparation of Samples for Analysis by SDS‐PAGE
  • Support Protocol 5: Solubility Analysis
  • Support Protocol 6: Preparation of Periplasmic Extracts
  • Support Protocol 7: Preparation of Extracellular Medium Samples
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Expression Under Control of pL Promoter: Temperature Induction

  Materials
  • Host E. coli strain (e.g., Coli B, W3110) transformed with plasmid containing gene for the protein of interest under control of p L promoter (see protocol 5)
  • LB medium (see recipe)
  • Antibiotic stock solution (see recipe)
  • 20‐ml sterile culture tube
  • 42°C shaking water bath
  • 150‐ml shaker flask, sterile

Basic Protocol 2: Expression Under Control of trp Promoter: Chemical Induction

  Materials
  • Host E. coli strain transformed with plasmid containing trp promoter and gene for the protein of interest (see protocol 5)
  • M9 medium (see recipe) with 0.5% (w/v) Casamino Acids (Difco)
  • Antibiotic stock solution (see recipe)
  • Indole‐3‐acrylic acid (IAA; see recipe)
  • 20‐ml sterile culture tube
  • 150‐ml shaker flask
  • 37°C orbital shaker

Alternate Protocol 1: Expression Under Control of lac/tac Promoters

  • Host E. coli strain containing lacI repressor gene and transformed with plasmid containing lac or tac promoter and gene for the protein of interest
  • Isopropyl‐β‐thiogalactopyranoside (IPTG; see recipe)
For this protocol, follow steps to of protocol 2 (trp promoter), substituting the appropriate host strain as the starting material. Replace step with the following and then return to steps and of protocol 2.

Alternate Protocol 2: T7 RNA Polymerase/Promoter Expression System

  • Host E. coli strain transformed with DNA encoding T7 RNA polymerase under control of lacUV5 promoter, plasmid containing p T7 and gene for the protein of interest, and (optional) plasmid containing gene for T7 lysozyme
  • Isopropyl‐β‐thiogalactopyranoside (IPTG; see recipe)
For this protocol, follow steps to of protocol 2, except begin with the appropriate transformed E. coli host strain, use ampicillin selection, and include a second antibiotic if the optional T7 lysozyme plasmid strategy is used (e.g., chloramphenicol at 30 mg/liter for pLysE or pLysL transformation). Replace step with the following and return to steps and of protocol 2.

Support Protocol 1: Transformation by Electroporation

  Materials
  • Host E. coli strain
  • LB medium (see recipe)
  • Water, ice‐cold
  • 10% (w/v) glycerol, ice‐cold
  • Plasmid DNA
  • SOC medium (see recipe)
  • LB plates with appropriate antibiotic (see recipe)
  • 1‐liter and 50‐ml centrifuge bottles, chilled
  • Centrifuge (e.g., Sorvall RC3B equipped with H6000A rotor, or equivalent), 4°C
  • 1.5‐ml sterile microcentrifuge tubes, chilled
  • Electroporation apparatus (e.g., E. coli Pulser Apparatus; Bio‐Rad)
  • Electroporation cuvettes, 0.2‐cm electrode gap, sterile and prechilled

Support Protocol 2: Plasmid Stability Analysis

  Materials
  • PBS ( appendix 2E)
  • Transformed E. coli host strain ( protocol 1Basic Protocols 1 and protocol 22, protocol 3Alternate Protocols 1 and protocol 42)
  • LB plates with and without antibiotic (see recipe)
  • 20‐ml sterile culture tubes
  • Sterile triangular glass rod
NOTE: Cells should be kept in an ice bath throughout the procedure to minimize alterations to the samples.

Support Protocol 3: Strain Storage

  Materials
  • Transformed E. coli host strain (see protocol 5)
  • LB medium (see recipe)
  • Antibiotic stock solution (see recipe)
  • 20% (w/v) glycerol, dispersed in sterile cryovials (see recipe)
  • LB plate (see recipe)
  • 20‐ml culture tube
  • 30° or 37°C orbital shaker

Support Protocol 4: Preparation of Samples for Analysis by SDS‐PAGE

  Materials
  • Sample of interest
  • 2× and 1× SDS sample buffer (unit 10.1)
  • Toothpicks, sterile
  • Sonicator (Branson, with microtip)

Support Protocol 5: Solubility Analysis

  Materials
  • Transformed E. coli host cells expressing the protein of interest
  • 25 mM HEPES (pH 7.6; autoclave and store up to 1 yr at 4°C)
  • 50‐ and 5‐ml centrifuge tubes
  • Centrifuge (e.g., Sorvall RC5B equipped with SS34 rotor), 4°C
  • Sonicator
NOTE: Cells should be kept in an ice bath throughout the procedure to minimize alterations to the samples.

Support Protocol 6: Preparation of Periplasmic Extracts

  Materials
  • Transformed E. coli cells expressing the protein of interest
  • PBS ( appendix 2E)
  • 20% (w/v) sucrose/ 10 mM Tris·Cl (pH 7.5), ice‐cold
  • 0.5 M EDTA (pH 8.0)
  • Ice‐cold water
  • Microcentrifuge, 4°C
NOTE: Cells should be kept in an ice bath throughout the procedure to minimize alterations to the samples.

Support Protocol 7: Preparation of Extracellular Medium Samples

  Materials
  • Transformed E. coli cells expressing the protein of interest
  • 2 g/liter BSA (optional; sterilize through a 0.22‐µm filter and store ≤1 month at 4°C)
  • 200 g/liter trichloroacetic acid (TCA; sterilize through a 0.22‐µm filter and store ≤1 month at 4°C)
  • 70% (v/v) ethanol
  • 0.22‐µm filters suitable for aqueous solutions
  • Microcentrifuge, 4°C
  • Ice bath
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Literature Cited

Literature Cited
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   Seidman, C.E., Struhl, K., and Sheen, J. 1989. Introduction of plasmid DNA into cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.8.1‐1.8.8. John Wiley & Sons, New York.
   Shatzman, A.R., Gross, M.S., and Rosenberg, M. 1990. Expression using vectors with phage λ regulatory sequences. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.3.1‐16.3.11. John Wiley & Sons, New York.
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Key References
   Goeddel, 1990. See above.
  Reviews most common expression systems in E. coli and provides a complete source of background information on the expression vectors as well as some hints on strategies for optimization.
   Hockney, 1994. See above.
  Comprehensive review on advances in protein expression in E. coli. Provides useful information regarding solubility, secretion, and fusions. Also discusses the difficult problem of expressing complex proteins such as multi‐transmembrane proteins in E. coli.
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