An Escherichia coli cell-free system for recombinant protein synthesis on a milligram scale

Methods Mol Biol. 2011;752:17-28. doi: 10.1007/978-1-60327-223-0_2.


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.

MeSH terms

  • Cell Extracts
  • Culture Techniques
  • DNA / genetics
  • Escherichia coli / cytology
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Plasmids / genetics
  • Protein Biosynthesis*
  • Protein Engineering / methods*
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Temperature
  • Time Factors


  • Cell Extracts
  • Recombinant Proteins
  • DNA