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Production of Eukaryotic Cell-Free Lysate from Leishmania tarentolae

  • Wayne A. Johnston
  • Kirill Alexandrov
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1118)

Abstract

In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathogenic to humans and does not require special laboratory precautions. An additional reason it is a convenient source organism for cell-free lysate production is that all endogenous protein expression can be suppressed by a single antisense oligonucleotide targeting splice leader sequence on the 5′-end of all protein coding RNAs. We describe simple procedures for cell disruption and lysate processing starting from bioreactor culture. We also describe introduction of genetic information via vectors containing species-independent translation initiation sites (SITS). We consider that such an inexpensive eukaryotic cell-free production system has many advantages when expressing multi-subunit proteins or difficult to express proteins.

Key words

Leishmania tarentolae Fluorophores Nitrogen cavitation Eukaryote Bioreactors SITS Overlap/extension PCR mCherry eGFP 

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Wayne A. Johnston
    • 1
  • Kirill Alexandrov
    • 1
  1. 1.Institute for Molecular BioscienceThe University of QueenslandSt. LuciaAustralia

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