LEXSY – Eukaryotic protein expression in Leishmania tarentolae
The unicellular kinetoplast protozoan Leishmania tarentolae, isolated from the Moorish gecko Tarentola mauritanica, not pathogenic to mammalians (Biosafety level 1) – was turned into the protein-producing host of our eukaryotic protein expression system LEXSY:
- eukaryotic host as easy to handle as E. coli: no specific labware, no cell biology equipment required
- fully eukaryotic protein expression machinery with post-translational modifications, including glycosylation and disulfide bond formation
- shuttle vectors: cloning in E. coli, expression in LEXSY host
- constitutive or inducible, intracellular or secretory expression of target proteins
- stable expression strains for constant protein production
Which LEXSY should I use?
In vivo or in vitro:
The LEXSY protein expression technology is available as live cell-based expression system (In Vivo LEXSY) and as cell-free translation system (In Vitro LEXSY).
- In Vivo LEXSY requires construction of an L. tarentolae expression strain that is suitable for fermentation in inexpensive media and that delivers high yields of recombinant proteins.
- In Vitro LEXSY allows protein production directly from a gene of interest (either as a PCR product or sub-cloned into an appropriate DNA vector) which enables ultrafast production of a large number of proteins in parallel but which is not suitable for infinite upscale.
|In Vivo LEXSY||In Vitro LEXSY|
Constitutive or inducible:
In Vivo LEXSY is available in two principle configurations that are constitutive or inducible.
- The constitutive system is the basic architecture which permits efficient production of a large variety of proteins. It is based on insertion of an expression cassette into the chromosomal ssu-locus. This cluster encodes the tandem 18S rRNA genes and is transcribed by the endogenous RNA Polymease I.
- The inducible system enables tight control of protein expression, analogous to the well-known bacterial T7 RNA Polymerase/TET repressor architecture. Expression is switched on by addition of an inducer (tetracycline) and thereby alleviates potential toxicity issues of an expressed target protein.
|Typical cultivation time||2-4 days||2-4 days||1-3 days||1-3 days|
|Number of available selection
- 4 alternative selection antibiotics available (Nourseothricin (NTC), LEXSY Hygro, LEXSY Bleo, LEXSY Neo)
- 2 alternative selection antibiotics available (LEXSY Bleo, LEXSY Neo)
Furthermore, the inducible LEXSY is available as integrative or episomal version. In the integrative version the expression cassette is stably inserted into the chromosomal ornithin decarboxylase (odc) locus whereas the episomal version makes use of amplification and oligomerisation of expression plasmids maintained extrachromosomally as self-replicating episomes in L. tarentolae cells.
Finally, the inducible configuration termed pLEXSY_I-blecherry enables efficient screening of high expression clones and online monitoring of induction using Cherry-fluorescence. This was achieved by fusion of the ble resistance and cherry fluorescence genes.
Manuals with description of LEXSY configurations and detailled protocols for cultivation, cloning, transfection, selection and evaluation of expression strains are available for download:
- Constitutive protein expression from genome integrated constructs
- Inducible protein expression from genome integrated constructs
- Inducible protein expression from episomal constructs
- In vitro expression from plasmid constructs
- In vitro expression from PCR products
LEXSY Conference Presentations
Recent presentations of LEXSY technology on international conferences are available:
- LEXSY talk at Halle Conference 2015
- LEXSY talk at FAP Meeting 2013 in Košice
- LEXSY talk at PepCon 2012 Beijing
- Talk at Nourseothricin Minisympoium Jena 2011
- Poster at Kinetoplastid Molecular and Cellular Biology Meeting (KMCBM) 2011 Woods Hole
- In Vitro LEXSY talk at Heart of Europe Conference (HEC) Schöneck 2010
Detailled protocols for cultivation, transfection, cryoconservation and handling of LEXSY are available:
- LEXSY cultivation tips n’tricks
- Low Voltage LEXSY standard electroporation protocol with BioRad Gene Pulser
- LEXSY electroporation protocol with multiporator
- publication describing Amaxa electroporation of l. tarentolae
- LEXSY clonal selection protocol
- protocol for preparation of LEXSY agar plates
- LEXSY polyclonal selection protocol
- overview of LEXSY transfection and selection
- LEXSY cryoconservation protocols