Leishmania tarentolae extract (LTE) Cell-Free Technology is a novel in vitro protein production system developed in the group of Prof. Kirill Alexandrov at the Institute for Molecular Biosciences (The University of Queensland). The system is based on the translation competent extracts of protozoan L. tarentolae.


 

Why Leishmania?

Leishmania is easy to grow in bioreactors like a prokaryote (such as E.coli), but is a non-pathogenic eukaryote with eukaryotic protein translation and folding machinery. Also, all endogenous message mRNAs in Leishmania carry an identical leader sequence, allowing a single antisense oligonucleotide to suppress translation of new unwanted endogenous Leishmania proteins in the lysate.
 

How it works:

The system is remarkably simple and allows single step production of recombinant protein from either plasmid or PCR-generated DNA templates. The system uses coupled transcription/translation; hence the lysate also contains enzymes and components necessary for conversion of introduced DNA into mRNA, which then codes for the desired protein.  LTE technology enables the production of difficult-to-express proteins rapidly and in a multiplexed format. PEF supplies bulk LTE lysate and provides template design and cloning services. Custom expression of genes in the cell-free system can also be performed.

 

pLTE Vectors:

The pLTE expression vectors are designed to be used in conjunction with the Leishmania tarentolae extract (LTE) in vitro translation system.

PEF offers 5 different vectors:

  1. pLTE (EGFP control)

  2. pLTE_6H_EGFP_3C

  3. pLTE_3C_EGFP_6H

  4. pLTE_6H_N and

  5. pLTE_6H_C

pLTE (EGFP control), pLTE_6H_EGFP_3C and pLTE_3C_EGFP contain an EGFP gene, which can be used as a positive control for transcription/translation reactions. pLTE_6H_N and pLTE_6H_C do not contain an EGFP gene. All vectors except the pLTE (EGFP control) contain a 6xHis-tag.

Additionally, all the vectors have a species independent translational sequence (SITS) incorporated upstream of the EGFP ORF. SITS consists of a poly(UUUUA)13 5′ UTR fused to a sequence which forms three mRNA stem-loop structures (Mureev et al., 2009). Translation of this sequence results in 17 additional amino acids at the N’ terminus of the target protein. This configuration ensures the highest yield of recombinant protein synthesis in the LTE and all other cell free expression systems.

You can choose to clone the gene of interest into the pLTE (EGFP control) vector via three cloning strategies:

  1. Replacement of EGFP control gene by target gene

  2. Fusion of target gene to N’ of EGFP gene

  3. Fusion of target gene to C’ of EGFP gene

Applications of LTE Expression System

The system is most generally suitable for applications where small amounts of difficult to express proteins (in other systems) are required.

  • Co-expression of multiple genes for production of protein complexes

  • Rapid protein engineering (truncations, insertions and site directed mutagenesis)

  • Construction of complete expressed proteomes

  • Functional deorphanization of genomes

  • Expression of difficult proteins (for instance malaria –derived proteins)

  • Expression of membrane proteins

  • Production of proteins for protein microarrays

Advantages of LTE Expression

  • Cost efficient high throughput expression

  • Eukaryotic protein folding

  • Low cost

  • Single tube format

  • Scalability

  • Effective incorporation of unnatural amino acids