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 5a2-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. © 2014 Springer Science+Business Media, LLC.
CITATION STYLE
Johnston, W. A., & Alexandrov, K. (2014). Production of eukaryotic cell-free lysate from leishmania tarentolae. Methods in Molecular Biology, 1118, 1–15. https://doi.org/10.1007/978-1-62703-782-2_1
Mendeley helps you to discover research relevant for your work.