LEXSY Expression

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

Using the Protozoan Leishmania tarentolae as a Protein Expression System

Talk held at HKI Colloquium in Jena, May 15, 2018

The inducible LEXSY 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 protein. Further, it was shown for a number of intracellular proteins that inducible expression achieves 5 – 10-fold higher yields than constitutive expression. Selection is performed with the antibiotics LEXSY Neo or LEXSY Bleo. Expression vectors with both alternative markers can be introduced into the same cell for co-expression of two proteins.

The 3rd generation of genome-integrated inducible LEXSY now allows convenient co-expression of the protein of interest with BleCherry conferring both, antibiotic resistance for selection with the antibiotic LEXSY Bleo as well as visible fluorescence under daylight as a direct read out for protein production.

Products & Ordering
LEXSinduce3 Expression Kit EGE-1410blecherry contains the integrative vector pLEXSY_I-blecherry3 for inducible cytosolic or secretory protein expression LEXSinduce3 Expression Kit EGE-1410ble contains the integrative vector pLEXSY_I-ble3 for inducible cytosolic or secretory protein expression LEXSinduce3 Expression Kit EGE-1410neo contains the integrative vector pLEXSY_I-neo3 for inducible cytosolic or secretory protein expression

Each kit contains:

  • pLEXSY_I expression vector of choice
  • pLEXSY_I-EGFP control vector
  • LEXSY host T7-TR as glycerol stocks
  • all components for preparation of 1 L LEXSY BHI medium
  • all selective antibiotics for 1 L medium
  • primer sets for insert sequencing and diagnostic PCR
  • an easy-to-follow manual, see PDF file in “Downloads”

All components of the kits are available also separately.

The episomal inducible 4th generation of LEXSY makes use of amplification and oligomerisation of expression plasmids maintained extrachromosomally as self-replicating episomes in the LEXSY host cells, providing enhanced number of target gene copies for transcription by T7 RNA polymerase.

It was shown for a number of proteins, that the episomal inducible expression achieved 5 – 10-fold higher yields than integrative inducible expression. High expression strains are obtained by clonal selection and screening which is based on fluorescence intensity resulting from the co-expressed BleCherry, egfp or Ds-red genes. Primary screening on solid media can be performed in daylight since the colours are visible by eye.

Products & Ordering
LEXSinduce4 Expression Kit EGE-1420blecherry contains the episomal vector pLEXSY_IE-blecherry4 for inducible cytosolic or secretory protein expression

Each kit contains:

  • pLEXSY_IE expression vector of choice
  • LEXSY host T7-TR as glycerol stocks
  • all components for preparation of 1 L LEXSY BHI medium
  • all selective antibiotics for 1 L medium
  • primer sets for insert sequencing
  • an easy-to-follow manual, see PDF file in “Downloads”

All components of the kit are available also separately.

The integrative constitutive LEXSY is the basic architecture which permits efficient production of a large variety of proteins. It is based on integration of an expression cassette into the chromosomal ssu-locus encoding the tandem 18S rRNA genes. This cluster is transcribed by the strong endogenous RNA Polymease I. Due to the expression profile of RNA Polymease I expression of target proteins parallels growth of the culture.

Dependent upon the cloning strategy target proteins are either steadily expressed in the cytoplasm of the host or secreted into the growth medium. Cytosolically expressed proteins can be isolated from easily disrupted cells, whereas secreted proteins are efficiently isolated from the medium by one-step affinity chromatography.

We recommend the standard kit with the vector pLEXSY-sat2.1 for selection of LEXSY expression strains with the antibiotic Nourseothricin (NTC).

Besides the sat marker, vectors with five other selection markers (ble, hyg, neo, bsd, pac) are available. Expression vectors with all six alternative markers can be introduced into the same cell for coexpression of up to six proteins.

Products & Ordering
LEXSYcon2.1 Expression Kit EGE-1310ble contains the integrative vector pLEXSY-ble2.1 for constitutive cytosolic or secretory protein expression LEXSYcon2.1 Expression Kit EGE-1310hyg contains the integrative vector pLEXSY-hyg2.1 for constitutive cytosolic or secretory protein expression LEXSYcon2.1 Expression Kit EGE-1310neo contains the integrative vector pLEXSY-neo2.1 for constitutive cytosolic or secretory protein expression
LEXSYcon2.1 Expression Kit EGE-1310sat contains the integrative vector pLEXSY-sat2.1 for constitutive cytosolic or secretory protein expression LEXSYcon2.1 Expression Kit EGE-1310bsd contains the integrative vector pLEXSY-bsd2.1 for constitutive cytosolic or secretory protein expression LEXSYcon2.1 Expression Kit EGE-1310pac contains the integrative vector pLEXSY-pac2.1 for constitutive cytosolic or secretory protein expression

Each kit contains:

  • pLEXSY-2.1 vector of choice
  • LEXSY host P10 as glycerol stocks
  • all components for preparation of 1 L LEXSY BHI medium
  • selective antibiotic for 1 L medium
  • primer sets for insert sequencing and diagnostic PCR
  • an easy-to-follow manual, see PDF file in “Downloads”


All components of the kits are available also separately.

The constitutive pLEXSY-2.1 vectors (EGE-271 to EGE-276) and inducible pLEXSY_I vectors (EGE-243 to EGE-251) were developed for both, intracellular or secretory expression of target proteins.

For constitutive expression we recommend the standard vector pLEXSY-sat2.1 for selection of LEXSY expression strains with the antibiotic Nourseothricin (NTC). For inducible expression we recommend the integrative vector pLEXSY_I-blecherry3 for selection of LEXSY expression strains with the antibiotic LEXSY Bleo and Cherry fluorescence monitoring of induction.

Products & Ordering
pLEXSY-ble2.1 EGE-271 Integrative constitutive expression vector Antibiotic selection of transfectants with LEXSY Bleo pLEXSY-hyg2.1 EGE-272 integrative constitutive expression vector antibiotic selection of transfectants with LEXSY Hygro
pLEXSY-neo2.1 EGE-273 Integrative constitutive expression vector Antibiotic selection of transfectants with LEXSY Neo pLEXSY-sat2.1 EGE-274 Integrative constitutive expression vector Antibiotic selection of transfectants with Nourseothricin (NTC, clonNAT)
pLEXSY-bsd2.1 EGE-275 Integrative constitutive expression vector Antibiotic selection of transfectants with LEXSY BSD pLEXSY-pac2.1 EGE-276 Integrative constitutive expression vector Antibiotic selection of transfectants with LEXSY Puro
pLEXSY_I-blecherry3 EGE-243 Integrative inducible expression vector Antibiotic selection of transfectants with LEXSY Bleo and expression monitoring with Cherry fluorescence pLEXSY_I-ble3 EGE-244 Integrative inducible expression vector Antibiotic selection of transfectants with LEXSY Bleo
pLEXSY_I-neo3 EGE-245 Integrative inducible expression vector Antibiotic selection of transfectants with LEXSY Neo pLEXSY_IE-blecherry4 EGE-255 episomal inducible bicistronic expression vector antibiotic selection of transfectants with LEXSY Bleo and expression monitoring with Cherry fluorescence

Control vectors and primers on request, email info@stratech.co.uk

The In Vitro LEXSY translation system is a NEW rapid, convenient, flexible and cost efficient tool for cell-free production of recombinant proteins for biochemical, biophysical and structural analysis.

The LEXSY cell extract for in vitro translation contains functional ribosomes and all essential components of the eukaryotic translation and folding machinery. For target mRNA generation the heterologous T7 RNA polymerase was added to the extracts. To ensure a low background the translation of endogenous host mRNAs is efficiently blocked by an antisense oligonucleotide making use of the unique gene organization of Leishmania.

DNA templates can be generated by cloning into a plasmid or by PCR amplification. The plasmid-based version was designed for high yield in vitro translation whereas the PCR-based version allows high-throuput applications.

Products & Ordering
LEXSY in vitro Translation Kit EGE-2002-15 for plasmid based cell-free protein synthesis LEXSY in vitro Translation Kit EGE-2010-15 for PCR based cell-free protein synthesis LEXSY in vitro Translation Cell Extract EGE-260 for cell-free protein synthesis

On request, custom cloning and expression of target genes can be performed at Jena Bioscience.

The following services are available:

  • custom cloning of target genes into a LEXSY expression vector of choice (including synthesis of LEXSY codon-optimized genes)
  • custom transfection and selection (polyclonal/clonal) of recombinant strains
  • evaluation of expression
  • expression optimisation
  • pilot affinity purification

Please contact us at technical@stratech.co.uk with questions or inquiries.

LEXSY host strains are available for the constitutive system (P10 strain) or the inducible system (T7TR strain). They are shipped without additional medium or culture flasks.

Products & Ordering
LEXSY host P10 LT-101 glycerol stocks for constitutive expression LEXSY host T7-TR LT-110 glycerol stocks for inducible expression

The LEXSY Cultivation Kits contain all components required for starting and establishing cultures of the two L. tarentolae strains LEXSY host P10 for constitutive expression or LEXSY host T7-TR for inducible expression in “ready-to-cultivate” formats.

Products & Ordering
LEXSY Cultivation Kit P10 LT-102 Contains LEXSY host P10 For constitutive expression LEXSY Cultivation Kit T7-TR LT-111 contains LEXSY host T7-TR for inducible expression

Each kit contains:

  • glycerol stocks of LEXSY host (upon request live suspension culture)
  • 50 ml ready-to-grow LEXSY BHI medium
  • all components for preparation of 1 L of LEXSY BHI medium
  • cell culture flasks for initial cultivation
  • cryo vials with glycerol for strain cryoconservation

LEXSY BHI cultivation media are based on brain-heart infusion and used for routine maintenance, evaluation, transfection and cryoconservation of LEXSY strains.

The complex cultivation media are avaiable as powder or liquid media kits.
All powder and liquid media kits are shipped with Hemin and Pen-Strep stock solutions.

Products & Ordering
LEXSY BHI – Liquid Media Kit ML-411 sterile, brain-heart infusion-based medium recommended for transfection and strain maintenance LEXSY BHI – Powder Media Kit ML-412 brain-heart infusion-based recommended for transfection and strain maintenance

Please note:

  • Hemin is essential for growth of LEXSY cultures.
  • Addition of Pen-Strep prevents growth of bacterial contaminations.
  • Following addition of these components the media can be used within two weeks.
    For specific instructions please, consult the particular media datasheets.
  • If the completed media are to be used after this period, appropriate amounts of additives have to be re-added.

The LEXSY Plating Kits are optimized for high efficiency of clonal selection of LEXSY expression strains. The kits contain all components of the medium (Agar base, buffered liquid medium, heat-inactivated fetal calf serum, hemin & Pen-Strep stock solutions). The comfort kit contains in addition plating spatula, 90 mm petri dishes, serological pipettes and – for convenient plating and inducible clone selection – nitrocellulose membranes. The basic kit does not contain fetal calf serum which must be provided by the user.


Products & Ordering
LEXSY Plating Kit comfort ML-451 components for solid medium brain-heart infusion- and fetal-calf-serum-based, with NC membranes, spatula, dishes and pipettes LEXSY Plating Kit core ML-452 components for solid medium brain-heart infusion- and fetal-calf-serum-based, without NC membranes, spatula, dishes & pipettes LEXSY Plating Kit basic ML-453 components for solid medium brain-heart infusion-based, without FCS, NC membranes, spatula, dishes & serological pipettes

For selection of recombinant LEXSY strains use our optimized LEXSY antibiotics:

  • Nourseothricin (NTC) the prime selection antibiotic for sat vectors
  • LEXSY Bleo for ble and blecherry vectors
  • LEXSY Hygro for hyg vectors
  • LEXSY Neo for neo vectors

Nourseothricin (NTC or clonNAT) is the prime selection antibiotic for recombinant LEXSY strains. It can efficiently be used also for other host systems as Gram-positive and Gram-negative bacteriaStreptomycetesyeastfilamentous fungiprotozoaplants etc.

Products & Ordering
Nourseothricin – Solution AB-101 NTC or clonNAT sterile ready-to-go stock solution Nourseothricin – Powder AB-102 NTC or clonNAT powder (non-sterile)
LEXSY Bleo AB-103 sterile ready-to-go stock solution LEXSY Hygro AB-104 sterile ready-to-go stock solution
LEXSY Neo AB-105 sterile ready-to-go stock solution LEXSY Tet AB-106 sterile ready-to-go stock solution (for inducible protein expression in LEXSY host T7-TR)
LEXSY BSD AB-107 sterile ready-to-go stock solution LEXSY Puro AB-108 sterile ready-to-go stock solution

Hemin and PenStrep stock solutions are used for complementation of synthetic and complex LEXSY cultivation media.


Products & Ordering
Pen-Strep ML-105 sterile 200x stock solution of penicillin and streptomycin Hemin (porcine) ML-108 sterile 500x stock solution in 30% triethanolamine

Please note:

  • Hemin is essential for growth of LEXSY cultures.
  • Addition of Pen-Strep prevents growth of bacterial contaminations.
  • Following addition of these components the media can be used within two weeks.
    For specific instructions please, consult the particular media datasheets.
  • If the completed media are to be used after this period, appropriate amounts of additives have to be re-added.

LEXSY Selection Guide: 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.

From gene to protein within approximately 6 weeksFrom gene to protein within 2 days or less
Scalable, suitable for production of large amounts of recombinant protein by cultivation in inexpensive mediaSmall scale protein preparation only
low costshigh costs

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.

ParametersConstitutive LEXSY LEXSYconInducible LEXSY LEXSinduce
Typical cultivation time2-4 days2-4 days1-3 days1-3 days
Number of available selection4(1)4(1)2(2)2(2)
(1) 4 alternative selection antibiotics available (Nourseothricin (NTC), LEXSY Hygro, LEXSY Bleo, LEXSY Neo)
(2) 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.

LEXSY in Parasitology

Leishmania tarentolae is a close relative of all pathogenic Leishmania species as well as of other pathogens such as Trypanosomes, Plasmodium and Toxoplasma. Due to this evolutionary proximity, the LEXSY technology is efficiently expressing parasite proteins with

  • High yields
  • Correct protein folding
  • Native post-translational modifications

LEXSY was used for overexpression and purification of functional parasite proteins. Insert A: Western blot of 93 kDa J-binding protein (JBP1) of Leishmania sp. Lane 1 = host control, lane 2 = induced culture, lane 3 = non-induced culture (Courtesy of S. Vainio, NCI Amsterdam). Insert B: Coomassie stain of immunoreactive surface proteins SAG1 (28 kDa) and SAG2 (15 kDa) of Toxoplasma gondii. Lanes 1 and 5 = host controls, lanes 2 and 3 = SAG1, lane 4 = SAG 2 secreted to the culture medium (Courtesy of M. Ebert, FZMB, Erfurt)

In addition, the expression vectors developed for LEXSY can be used for creation of transgenic strains of other Leishmania species including L. amazonensis, L. donovani, L. infantum, L. major, L. mexicana and also Crithidia sp. as well as the plant parasite Phytomonas serpens. These features of LEXSY enable functional characterization of parasite proteins, investigation of parasite-host interactions, in vivo and in vitro screening of anti-leishmanial drugs and vaccine development.

A: Expression and functional analysis of the catalytic domain of α-N-acetylglucosaminyltransferase from Trypanosoma cruzi (TcOGNT2cat) in LEXSY by Western blotting (left) and enzymatic activities (right). P10 = non-transfected host strain; wt = P10 expressing wild-type TcOGNT2cat; D234A and D234N = single point mutants (from Heise et al. 2009). B: Subcellular localization of ferrous iron transporter LIT1 expressed in L. amazonensis Δlit promastigotes using pLEXSY constructs. Immunofluorescence demonstrated different targeting of wild type and mutant proteins to the plasma membrane. LIT1 immunofluorescence = green, parasite DNA = blue, FITC = anti-LIT1 IF on fixed/non-permeabilized promastigotes, FITC-LIVE = anti-LIT1 IF on live promastigotes (from Jacques et al. 2010). C: EGFP imaging in L. major reporter strain stably transfected with pLEXSY-egfp construct by Epi-fluorescence microscopy of recombinant L. major promastigotes (left) and intracellular amastigotes in bone marrow-derived macrophages (right) (from Bolhassani et al. 2011). D: Expression of protozoon RabGTPases originating from L. tarentolae or P. falciparum in PCR-based In Vitro LEXSY. Coomassie stained SDS-PAGE gel loaded with EGFP-Rab GTPases eluted from a GFP binding matrix. For details see In Vitro LEXSY manual (adapted from Kovtun et al. 2010).

Click here to view the Reference listing LEXSY in parasitology 

Structural Biology: LEXSY Proteins for NMR and X-ray Crystallography

The applicability of LEXSY for structural biology was demonstrated by successful 15N-HSQC NMR analysis of a 28 kDa 15N-Val labeled protein purified from recombinant LEXSY strain grown in a synthetic LEXSY cultivation medium. All 18 Val residues of in vivo labeled protein could be completely assigned in 15N-HSQC NMR spectrum in full agreement with X-ray crystallography (Niculae et al. 2006). Since Leishmania tarentolae is auxotrophic for 11 amino acids and can be grown in chemically defined media multiple options for labeling strategies are offered. Alternatively to chemically defined media labeling strategies in complex media were developed (Foldynová-Trantírková et al. 2009).


Click here for Reference publication Niculae et al. (2006)

Click here for Reference publication Foldynova-Trantirkova et al. (2009)


15N-HSQC NMR analysis of 15N-Val labeled EGFP purified from recombinant LEXSY strain. For detailed description refer to Niculae et al. (2006).

It was shown, that LEXSY-expressed proteins can be subjected successfully to crystallography and X-ray analysis. The resolution of a new protein structure was achieved for LEXSY expressed hu Cu/Zn superoxide dismutase SOD1 (Gazdak et al. 2010).

Click here for Reference publication Gazdog et al. (2020)

Structure determination of the new P212121 crystal form of LEXSY-produced human Cu/Zn superoxide dismutase (SOD1). The asymmetric unit contains six SOD dimers arranged as two triangular wheels around sulfate ions. The wheels are arranged in a side-to-side fashion (Gazdag et al. 2010).

Another example of a LEXSY produced protein which was successfully crystallized is the cysteine proteinase legumain (Dall & Brandstetter 2012). Screening for crystallization conditions of legumain has been carried out with the JBScreen Classic kit. Super-activated legumain was successfully crystallized in complex with a specific inhibitor and using the heavy-atom soaking method, and mercury-derivative crystals were obtained. Single anomalous scattering dataset was collected to a resolution of 2.47 Å and the structure determined with a combination of isomorphous and anomalous scattering. The preliminary X-ray diffraction data analysis of active legumain has been presented and the structure of legumain glycoforms will be published elsewere (Hans Brandstetter, personal communication).


Click here for Reference publication Dall & Brandsetter (2012)


Crystals of mature legumain produced in LEXSY and X-ray diffraction image (from Dall & Brandstetter 2012).

LEXSY: Expression of recombinant Antibodies

Recombinant production of antibodies with focus on monoclonal antibodies (MAbs) has become a challenging task due to the rapidly expanding pharmaceutical and diagnostic markets.

LEXSY was evaluated for production of heavy and light chains of human IgG, single chain antibodies and Fc fusions. Recombinant Fc fusions were efficiently expressed in LEXSY, completely secreted to the culture medium and one-step affinity purified with Protein A sepharose with yields of ca. 10 mg/L. SDS PAGE analysis demonstrated that the proteins were secreted in the native configuration as dimers (JBS not published).

Purification of Fc fusion protein from LEXSY cultivation medium. Lane 1 molecular size marker, 2-3 Protein A sepharose-purified Fc fusion under reducing conditions, 4 dto. non-reducing conditions (JBS not published).

LEXSY: Expression of complex oligomeric Proteins

Many proteins of higher organisms are oligomers consisting of more than one polypeptide chain, and recombinant production of these complexes in an active form often requires simultaneous co-expression of the individual polypeptides. LEXSY allows up to four different antibiotic selection markers that can be used for expression of up to four different proteins simultaneously facilitating production of functional oligomers.

LEXSY was used to express human laminin-332 (α3β3γ2), a large heterotrimeric glycoprotein and essential component of epithelial basal lamina that promotes cell adhesion and migration (Phan et al. 2009).

Click here for Reference publication

Model of heterotrimeric laminin-332 (left) and Western blot of purified 420 kDa laminin heterotrimer separated under non-reducing conditions. Lanes 1 molecular size marker (kDa), 2 laminin from 293-F cells (2 forms), 3 laminin from LEXSY (one defined form) after Phan et al. (2009).

Alternatively, avoiding limitation by availability of selection markers in vivo, oligomeric proteins can be obtained using In Vitro LEXSY by co-expession of the respective polypeptides in the same extract.


LEXSY: Mammalian-type Glycosylation

Glycosylation is a major posttranslational modification of a large variety of secreted and membrane proteins and is often a pivotal factor for folding, function and stability. Due to the absence of glycosylation pathways in prokaryotes, recombinant glycosylated proteins cannot be produced in e.g. bacteria. Further, glycosylation in most alternative eukaryotic expression hosts such as yeast and insect differs largely from the desired mammalian-type glycosylation. Despite several improvements including glycoengineering have been reported for these two systems, an expression system with adequate mammalian-type glycosylation is still highly desirable for protein expression in research, diagnostics and pharmaceutical applications.

Glycosylation in LEXSY was thoroughly investigated using recombinant human erythropoietin (EPO) as a model. EPO expressed in LEXSY was shown to be efficiently secreted into the culture medium, natively processed at the N-terminus and fully biologically active. Glycosylation analysis revealed two glycans, a complex mammalian-type biantennary oligosaccharide and the Man3GlcNAc2 core structure (Breitling et al. 2002). LEXSY is thus the first biotechnologically useful unicellular eukaryotic system producing biantennary fully galactosylated, core α-1,6-fucosylated N-glycans. In addition, the N-glycosylation pattern was exceptionally homogenous consisting of only two defined glycoforms. This glycosylation pattern was also found in LEXSY expressed human interferon γ; and host gp63 surface glycoprotein. While glycoproteins from other eukaryotes are typically heterogenous multi-glycoform populations, LEXSY-derived proteins are expected to be prone to crystallization and subsequent structure determination.

Click here for Reference publication


Analysis of recombinant human Erythropoietin (EPO) isolated from culture supernatants of a LEXSY expression strain. A: Western blot of commercial recombinant human EPO produced in CHO cells (1) and LEXSY secreted EPO before (2) and following (3) de-glycosylation with N-glycosidase F (PNG). B: Electrophoretic resolution of heterogenous population of CHO-derived EPO. Glycan structures are depicted at left. C: Enzymatic resolution of complex and core glycan structures released from LEXSY-produced EPO (for details refer to Breitling et al. 2002).

Solubility and Functionality of expressed Proteins

Incorrect folding and insufficient solubility – resulting in compromised biological activity – are the major shortcomings of prokaryotic expression systems. Due to LEXSY’s fully eukaryotic protein synthesis/folding/modification machinery most proteins of higher organisms expressed in LEXSY are correctly folded and processed and therefore, are obtained in a fully functional state.

Selected examples of LEXSY-expressed proteins with full biologic activity:


Protein Localisat. Origin Reference
Erythropoietin sec human Breitling et al. 2002
Surface Antigen 1& 2 sec Toxoplasma gondii Ebert et al. 2007 not publ.
Proprotein Convertase 4 sec rat Basak et al. 2008
Laminin-332 sec human Phan et al. 2009
Cu/Zn superoxide dismutase cyt human Gazdag et al. 2010
Tissue Plasminogen Activator sec human Hemayatkar et al. 2010
N-Acetyl Serotonin Methyl Transferase (ASMT) cyt human Ben-Abdallah et al. 2010
Hydroxynitrile Lyase (MeHNL) cyt cassava plant Dadashipour et al. 2011
Coagulation factor VII cyt human Mirzaahmadi et al. 2011








LEXSY Biosafety Status

Leishmania tarentolae is a unicellular, flagellated protozoan organism and belongs to a family of parasites shuttling between sandflies and vertebrates. Since isolation from its lizard host Tarentola mauretanica in 1921, L. tarentolae was kept in axenic culture. It is not pathogenic to mammalians and is fully approved for use in biosafety level 1 (S1) laboratories. Genome sequencing of L. tarentolae revealed loss of genes asscciated to the intracellular stage of human pathogenic species (Raymond et. al 2012).

Leishmania drawing from: fundacionio.org/img/parasitology/leishmania.html


The Leishmania Protein Expression System (LEXSY) can be used without a license in any not-for-profit research in academic or public institutions.

The use of LEXSY and its components for all commercial purposes, however, requires a separate license from Jena Bioscience. Commercial use includes but is not limited to:

  • the use of any protein or other substance produced by LEXSY as reagents in screening to discover and/or promote candidate compounds for sale to a customer, distributor, wholesaler or other end user in therapeutic, diagnostic, prophylactic, and/or veterinary areas.
  • the manufacture, sale or offer to sell of any product containing proteins or other substances produced by LEXSY.
  • the production of recombinant protein pharmaceuticals.
  • “Contract research” to any third party or “Contract manufacturing” for any third party that has not been granted a license to use LEXSY.

For further information and for terms of licensing: technical@stratech.co.uk

Using the Protozoan Leishmania tarentolae as a Protein Expression System Talk held at HKI Colloquium in Jena, May 15, 2018

LEXSY taking off: Selected examples for protein production with Leishmania tarentolae Talk held at 5th Halle Conference, February 20, 2015

From gene to crystallization within two days – LEXSY cell-free protein production Talk held at HEC 13, September 24, 2010

Q1: During construction of Leishmania expression plasmid in E. coli I encountered structural plasmid instability. How can I avoid this problem?

A1: LEXSY expression vectors contain repetitive and homopolymeric DNA stretches within the non-translated regions (utr). Therefore, it is recommended to choose recipient E. coli strains usually tolerating these motives such as XL-10, Stbl2 or Stbl4, DH5α, SURE or related and to incubate them at 30°C (see LEXSY manuals chapter 4.2). If the particular vector/insert combination with your target gene is not tolerated in one of these strains (indicated by plasmid deletions) try another recipient mentioned, rather than analyzing further clones or to repeat transformation with the same recipient strain.

Q2: Can the target gene be introduced into L. tarentolae on a circular plasmid?

A2: Yes, circular plasmids will be maintained in L. tarentolae cells extrachromosomally. For high-yield protein expression from episomal constructs we developed the 4th generation of inducible LEXSY (EGE-1420). However, if integrative expression plasmids are transfected in circular form, the yields of expressed proteins are significantly reduced (to 20-40%) compared to chromosomal integration of a single copy of this target gene. Therefore, it is important to completely linearize the integrative expression plasmids or to transfect the cells with gel-isolated fragments (see LEXSY manuals chapter 4.3).

Q3: There are four different antibiotic resistance markers available for the constitutive expression system. Which marker marker should I prefer?

A3: We recommend to get started with Nourseothricin (NTC) (Nourseothricin) selection using the sat vectors, since establishing recombinant strains is most straightforward with this antibiotic. The order of selection antibiotics regarding their efficiency of eliminating non-recombinant background is: Nourseothricin (NTC) > LEXSY Hygro > LEXSY Bleo > LEXSY Neo.

Q4: Is it possible to co-express two secreted proteins? Would this involve inserting multiple cDNAs into a single expression plasmid or using multiple plasmids with different antibiotic selection markers?

A4: It is possible to co-express up to four secreted or intracellular proteins in the same cell since we offer expression vectors with four alternative antibiotic selection markers and the ssu integration site is present in multiple copies per genome whereupon inactivation of up to four copies does not influence host physiology. Co-expression of up to four gene copies of cytosolic EGFP, superoxide dismutase (SOD1) or secretory erythropoietin (EPO) as well as of a secretory laminin heterotrimer were demonstrated successfully (Jena Bioscience, not published; Gazdag et al. 2010Phan et al. 2009). Please, refer also to A3 & A8.

Co-expression of multiple target genes from the same vector requires introduction of functional intergenic regions in between the ORFs for proper post-transcriptional processing. This alternative approach was successfully demonstrated for over-expression of human tissue plasminogen activator t-PA (Hemayatkar et al. 2010).

Q5: Can Leishmania be transfected by methods other than electroporation?

A5: Except electroporation, only particle bombardment was described for transfection of Leishmania (Sbicego et al. 1998). All attempts to use chemical transfection methods were up to date unsuccessful (Jena Bioscience, not published).

Q6: How long does it take to get the recombinant Leishmania strain after transfection?

A6: Polyclonal selection of recombinant cell lines in suspension cultures takes routinely two weeks. Within approximately seven days after electroporation differences between the cells electroporated under the same conditions with your target gene or without DNA (negative control) are becoming visible by microscopy. After this it takes another week following dilution into fresh growth medium with selection antibiotic to discriminate visually between the turbid recombinant culture and the clear negative control. However, under certain circumstances more time may be needed for additional 1:10 dilutions (see LEXSY manual for constitutive expression chapter 5.2).
Clonal selection on BHI agar requires about the same time. Colonies start to appear 5-7 days after plating. Approximately 7-9 days after plating they can be transferred into 24 well format and it takes two more days for expansion into 10 ml cultures for further analysis.

Q7: Are all cells of a stable transfected L. tarentolae line considered to harbor the genomic integration of the transfected expression cassette after selection?

A7: Yes, we showed by fluorescence microscopy, that virtually all cells of a stable LEXSY line selected in suspension culture expressed the transfected egfp gene (see Figure 1 in the LEXSY brochure 2011).

Q8: What are the relative advantages and disadvantages of constitutive versus inducible expression in LEXSY? Will constitutive expression give us high yields?

A8: The features of constitutive and inducible LEXSY configurations are compared in the LEXSY Configurations section of the JBS website and in the LEXSY Brochure 2011 pages 7-10. The inducible LEXSY is recommended for target proteins, which are potentially toxic for the expression host (e. g. proteases, nucleases, kinases). However, this configuration allows co-expression of only two target proteins, whereas with the constitutive LEXSY four target proteins can be co-expressed in the same cell.
Concerning the yields it was found with several cytosolic proteins that the yields in the inducible configuration were 5-10x higher as compared to the constitutive, whereas in case of several secretory proteins the yields were comparable in both configurations.

Q9: Does codon usage bias play a role in LEXSY and do you recommend using a synthetic gene optimized for the Leishmania codon usage? Our target genes are AT rich.

A9: Yes, whenever your budget allows, we recommend gene synthesis with L. tarentolae optimized codon usage because several synthetic genes were expressed at high levels. However, these findings are case sensitive since there are also numerous examples of highly expressed genes with native (e.g. human) codon bias and using synthetic genes does not guarantee higher expression levels than using the corresponding native genes.
Also, codon optimized gene synthesis changes the mRNA sequence and possibly its stability which may contribute to the final target protein yield. In one case we showed, that enhanced mRNA stability was indeed the reason for (or at least significantly contributed to) higher expression levels of a synthetic gene versus the native variant. To answer this question for your particular gene you should analyze expression at mRNA and protein levels.

Q10: What are the optimal conditions for expression of my recombinant target protein in LEXSY?

A10: The optimal conditions for expression of target proteins in LEXSY should be determined individually. Recommended are time course experiments under variable growth conditions such as static or agitated suspension cultures. We found e.g. that secreted proteins could be obtained at higher yields from agitated suspension cultures.
In case of constitutive expression you may also consider, that the ssu locus – the integration site of target genes – is transcribed at highest rates during the growth phase of the cultures. For inducible LEXSY, variation of time point of induction and of inducer concentration may influence target protein yields (see inducible LEXSY manuals chapter 8.6). If the target proteins are stable and non-toxic to the host, induction at inoculation usually results in the best yields.

Q11: Can target proteins be purified with affinity tags from LEXSY?

A11: Yes, affinity purification with His- or Strep-tags worked well in LEXSY with Strep-tags appearing to be more specific than His-tags. The LEXSY vectors are provided with the option for fusion of the target protein sequence to a C’ hexahistidine tag. Other short tag sequences can be added directly to the gene of interest before insertion into the expression vector. This way additional amino acids resulting from vector sequences (polylinker) can be avoided.

Q12: A key problem in protein expression in E. coli is the insolubility of the recombinant proteins. Are the proteins expressed in LEXSY soluble and did you observe inclusion bodies?

A12: LEXSY has the advantage of a fully functional eukaryotic protein biosynthesis, folding and modification machinery. A eukaryotic – especially mammalian – target protein is expected to fold more natively in Leishmania than in E. coli. All proteins we tested so far were soluble and proteins insoluble in E. coli were expressed in soluble form in LEXSY (for examples see p.18 of the LEXSY brochure 2011 and the chapter “Selected Applications” of the LEXSY section of the Jena Bioscience website). In addition to folding, LEXSY performs mammalian-type posttranslational modifications as N-glycosylation which may be essential for activity, function and stability of the target protein.

  • L. tarentolae needs aerobic conditions. The strains can be maintained as continuous suspension culture with regular dilutions (see chapter 3.2 of LEXSY manuals). All cultivations are performed at 26°C. Higher temperatures lower the growth-rates and vitality significantly and L. tarentolae will not survive at 37°C


  • All growth media should be supplemented with Hemin (Cat. No ML-108) which is essential for Leishmania. Hemin is light-sensitive, so Leishmania must be cultivated in the dark. After completion with Hemin the medium must be stored in the dark at 4°C. For optimal growth and vitality the completed medium should be used within 2 weeks. However, if this shelf live is exceeded, it is possible to re-add Hemin (and PenStrep) and to use this medium for 2 more weeks


  • For maintaining LEXSY strains for transfection and analysis it is convenient to grow static suspension cultures in 10 ml LEXSY BHI medium (Cat. No. ML-411, 412) in ventilated tissue culture (TC) flasks. Don´t use agitated cultures for strain maintenance since cells will age much faster. It is not necessary or growth-promoting, to add serum to the BHI medium


  • Best results are obtained with inoculations during early stationary phase. Avoid repeated successive dilution of cultures of low cell densities as this may drop growth. However, occasional higher dilutions of stationary cells at e.g. 1:100 will not adversely affect subsequent growth. It is convenient to dilute 10 ml cultures 1:50 on Monday and 1:20 on Friday and to incubate TC flask upright, lowering aeration for longer intervals between passages. Don’t cultivate Leishmania much longer than for 7 days in the same medium without dilution. For cultivation for transfection see chapter 4.4, for cultivation for protein expression see chapter 5.4 of the LEXSY manuals


  • Always control appearance and motility of the cells by microscopy. Cells of mid-growth phase cultures are of drop-like shape, approx. 15×5 µm in size with one flagellum at the flat end, and motile. These cells are most efficient for transfection and plating on solid media. Mid-growth phase cultures always contain subpopulations of non- or less motile cells and of cells of different shape. Don’t hesitate to transfect, plate or preserve a culture with drop-like cells containing such subpopulations. Cells of older cultures get longer and thinner (needle-like shape) and remain motile. Enhanced motility may result from nutrient deprivation or other limitations and must not necessarily be a sign of mid growth culture stage. Also, bacterial, fungal or other contaminations may be identified by microscopy


  • Keep patient, esp. if you are used to working with bacteria. Leishmania cells are protozoans with regular doubling times of 7 h in static suspension cultures and 4-5 h in agitated cultures. They need their time to grow or to adapt to new conditions


  • If you – despite following these instructions – encounter growth problems with the host strain, sediment cells 3 min at 2000g, resuspend pellet carefully in fresh growth medium and continue incubation in ventilated TC flasks. This approach was very helpful in rescuing cultures esp. after transfection


  • Don’t centrifuge Leishmania cultures at high speed >10.000g and don’t resuspend cell pellets by rigorous vortexing. The cells are sensitive to these procedures and may lyse. Centrifugation at 2000-3000g is sufficient for sedimentation and makes gentle and quick resuspension of cell pellets easier. If required, prolong centrifugation time rather than using higher speed


  • If you cultivate LEXSY strains in bioreactors be careful with stirring. We found it sufficient to aerate the culture in a 10 L fermentation without stirring for obtaining high cell densities up to 109 cells/ml. If you intend to use a stirrer, avoid high sharing forces


outstanding technical support


we offer a full product guarantee


we offer free delivery to UK universities and non profit organisations