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.

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.

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.

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.

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.

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 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.

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 bacteria, Streptomycetes, yeast, filamentous fungi, protozoa, plants etc.

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.

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.

(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.

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).

 

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

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. 2010; Phan 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