Unit

UNIT 6.3 Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

  1. Ira Palmer,
  2. Paul T. Wingfield

Published Online: 1 NOV 2012

DOI: 10.1002/0471140864.ps0603s70

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Palmer, I. and Wingfield, P. T. 2012. Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli. Current Protocols in Protein Science. 70:6.3:6.3.1–6.3.20.

Author Information

  1. National Institutes of Health, Bethesda, Maryland

Publication History

  1. Published Online: 1 NOV 2012
  2. Published Print: NOV 2012

Abstract

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low-speed centrifugation. Following pre-extaction (or washing), protein is extracted from washed pellets using guanidine⋅HCl. The solubilized and unfolded protein is either directly folded or further purified by gel filtration in the presence of guanidine⋅HCl as described in this unit. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS-PAGE. Curr. Protoc. Protein Sci. 70:6.3.1-6.3.20. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • recombinant protein;
  • Escherichia coli;
  • inclusion bodies;
  • protein extraction;
  • protein solubilization;
  • guanidine⋅HCl;
  • protein purification;
  • cell lysis