Use of the SHuffle Strains in Production of Proteins

Guoping Ren1, Na Ke1, Mehmet Berkmen1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.26
DOI:  10.1002/cpps.11
Online Posting Date:  August, 2016
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Abstract

Escherichia coli continues to be a popular expression host for the production of proteins, yet successful recombinant expression of active proteins to high yields remains a trial and error process. This is mainly due to decoupling of the folding factors of a protein from its native host, when expressed recombinantly in E. coli. Failure to fold could be due to many reasons but is often due to lack of post‐translational modifications that are absent in E. coli. One such post‐translational modification is the formation of disulfide bonds, a common feature of secreted proteins. The genetically engineered SHuffle cells offer an expression solution to proteins that require disulfide bonds for their folding and activity. The purpose of this protocol unit is to familiarize the researcher with the biology of SHuffle cells and guide the experimental design in order to optimize and increase the chances of successful expression of their desired protein of choice. Example of the expression and purification of a model disulfide‐bonded protein DsbC is described in detail. © 2016 by John Wiley & Sons, Inc.

Keywords: shuffle cytoplasm; DsbC; Disulfide bonded protein production; E. coli

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expression of Disulfide Bonded Proteins in SHuffle Cytoplasm
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

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Internet Resources
  https://www.youtube.com/watch?v=JYpGZE‐qHwk
  Video that explains the nature of a disulfide bond.
  https://www.youtube.com/watch?v=AyjLWVC2fHo
  Compartmentalization of disulfide bond formation.
  https://www.youtube.com/watch?v=46kHpgPa5W8
  Video that explains disulfide bond formation in the periplasm.
  https://www.youtube.com/watch?v=ZZmR17j4enk)
  Video that explains disulfide bond formation in the cytoplasm of SHuffle.
  https://www.neb.com/products/protein‐expression‐and‐purification‐technologies/e‐coli/shuffle‐strains‐for‐the‐expression‐of‐multi‐disulfide‐bonded‐and‐difficult‐to‐express‐proteins/how‐to‐use‐shuffle)
  A brief video guide on how to use SHuffle.
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