An Escherichia coli Cell-Free System for Recombinant Protein Synthesis on a Milligram Scale

  • Luke A. MilesEmail author
  • Gabriela A. N. Crespi
  • Sen Han
  • Andrew F. Hill
  • Michael W. Parker
Part of the Methods in Molecular Biology book series (MIMB, volume 752)


In vitro translation systems derived from a wide range of organisms have been described in the literature and are widely used in biomedical research laboratories. Perhaps the most robust and efficient of these cell-free systems is that derived from Escherichia coli. Over the past decade or so, experimental strategies have been developed which have enhanced the efficiency and stability of E. coli cell-free systems such that we can now prepare recombinant proteins on a scale suitable for purification and analysis by biophysical and structural biology techniques, which commonly require relatively large quantities of protein. This chapter describes in detail the protocols employed in our laboratory to prepare translationally active E. coli extracts and to synthesise proteins on a milligram scale from these extracts.

Key words

Cell-free protein synthesis In vitro translation Escherichia coli S30 


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Luke A. Miles
    • 1
    Email author
  • Gabriela A. N. Crespi
  • Sen Han
  • Andrew F. Hill
  • Michael W. Parker
  1. 1.St. Vincent’s Institute of Medical ResearchFitzroyAustralia

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