Strategies to Optimize Protein Expression in E. coli

Dana M. Francis1, Rebecca Page1

1 Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.24
DOI:  10.1002/0471140864.ps0524s61
Online Posting Date:  August, 2010
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Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post‐translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli. Curr. Protoc. Protein Sci. 61:5.24.1‐5.24.29. © 2010 by John Wiley & Sons, Inc.

Keywords: protein expression; E. coli; fusion proteins; proteases; heterologous protein; purification tags; expression tags; expression strains and vectors; folded protein; active protein

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Table of Contents

  • Introduction
  • I. Representative Protocol for Expressing Proteins in Bacteria
  • II. Properties of the Gene and Protein that Influence Expression and Solubility
  • III. Properties of the Vector that Influence Expression and Solubility
  • IV. E. coli Host Strains that aid Expression of Heterologous Proteins
  • V. The Solubility of Proteins can be Improved by Changing Expression Conditions
  • VI. Enhancing Solubility by Coexpression with Other Proteins
  • Anticipated Results and time Considerations
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
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