Species-independent translational leaders facilitate cell-free expression

Nat Biotechnol. 2009 Aug;27(8):747-52. doi: 10.1038/nbt.1556. Epub 2009 Aug 2.


Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems differ substantially from each other with respect to efficiency, scalability and the ability to produce functional eukaryotic proteins. Here we describe species-independent translational sequences (SITS) that mediate efficient cell-free protein synthesis in multiple prokaryotic and eukaryotic systems, presumably through bypassing the early translation initiation factors. We use these leaders in combination with targeted suppression of the endogenous Leishmania tarentolae mRNAs to create a cell-free system based on this protozoan. The system can be directly programmed with unpurified PCR products, enabling rapid generation of large protein libraries and protein variants. L. tarentolae extract can produce up to 300 microg/ml of recombinant protein in 2 h. We further demonstrate that protein-protein and protein-small molecule interactions can be quantitatively analyzed directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions / genetics*
  • Animals
  • Base Sequence
  • Cell Extracts
  • Cell-Free System
  • Leishmania / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Biosynthesis*
  • Species Specificity
  • Spectrometry, Fluorescence


  • 5' Untranslated Regions
  • Cell Extracts