Overview of the Purification of Recombinant Proteins

Paul T. Wingfield1

1 Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.1
DOI:  10.1002/0471140864.ps0601s80
Online Posting Date:  April, 2015
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When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self‐cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell‐breakage methods, protein purification methods and strategies are essentially the same. © 2015 by John Wiley & Sons, Inc.

Keywords: recombinant protein; protein purification; protein folding; protein expression; protein purification strategies

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Table of Contents

  • Protein Expression
  • An Overall Summary of Protein Production and Characterization
  • Protein Sequence and Compositional Analysis
  • Characteristics of the Host‐Vector System
  • Solubility and Location of the Protein
  • Strategies for Isolation of Soluble Proteins
  • Strategies for Isolation of Insoluble Proteins
  • Expression of Glycoproteins
  • Strategies for Isolation of Membrane Proteins
  • Some Examples of Protein Expression and Purification
  • Protein Handling
  • Scale of Operations and Aims of Purification
  • Specialized Equipment
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

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