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   LEXSY Technology – a valuable tool for Kinetoplastida research
   
   


* In this issue:

(1) Introduction
(2) Expression of parasite proteins using LEXSY
(3) Construction of transgenic Leishmania reporter strains
(4) LEXSY products – all you need for cloning, expression, strain selection, and cultivation

   
       
  
   
   

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    (1) Introduction
   


Based on the non-pathogenic S1 laboratory strain Leishmania tarentolae – a parasite of lizards – we have developed the protein expression platform LEXSY that allows production of a wide range of recombinant proteins

as well as Due to the similarity of LEXSY's protein folding and modification machinery to that of other Kinetoplastidae species (e.g. pathogenic Leishmania or Trypanosoma), LEXSY is perfectly suitable for efficient production of large amounts of proteins of quite a few notorious human parasites (Figure 1A).

In addition, the expression vectors developed for LEXSY (pLEXSY family) can be used for creation of transgenic Leishmania species including L. amazonensis, L. donovani, L. infantum, L. major, L. mexicana and also the plant parasite Phytomonas serpens (Figure 1B-C).

These features enable functional characterization of parasite proteins, investigation of parasite-host interactions, in vivo and in vitro screening of anti-leishmanial drugs and vaccine development.

Figure 1

(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 Δlit1 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 GFP-Cap matrix. For details see In Vitro LEXSY manual (adapted from Kovtun et al. 2010).


   
    (2) Expression of parasite proteins using LEXSY
   


Heise et al. expressed α-N-acetylglucosaminyltransferase from Trypanosoma cruzi in L. tarentolae using pLEXSY vectors carrying sequences for full length intracellular protein and for secretory wild type or mutant catalytic ectodomains (Figure 1A).

Jacques et al. functionally characterized the ferrous ion membrane transporter LIT1 expressed from a pLEXSY construct in a Δlit1 strain of L. amazonensis by sub-cellular localization in parasite cells (Figure 1B), intracellular growth rates of parasites in macrophages, and infection of mice.

Smirlis et al. investigated the interaction of Ran-GTPase with histones and its sub-cellular localization in a L. donovani strain harbouring a pLEXSY-LdRan episome.

Xingi et al. validated the enzyme glycogen synthase kinase-3 (GSK-3) as potential drug target by expression in L. donovani harbouring a pLEXSY construct and characterized the purified enzyme biochemically.

Bhattacharya et al. used the LEXSY T7-TR strain (expressing T7 RNA polymerase and TET repressor) for knock-down experiments with a cytosolic cAMP phosphodiesterase of L. donovani (LdPDEA) associated with resistance of parasites to macrophage oxidative damage.


   
    (3) Construction of transgenic Leishmania reporter strains
   


Lang et al. described the generation of a L. amazonensis strain stably expressing firefly luciferase from a pLEXSY construct. This strain was used for in vitro studies with parasite-transfected macrophages and for development of an in vivo model, allowing sensitive and rapid non-invasive real time bioluminescence imaging of parasites in living animals.

Lecoeur et al. employed the same approach for development of a mouse model for cutaneous Leishmaniasis caused by L. major and for evaluation of a topical therapy with a third-generation aminoglycoside ointment.

Barak et al. used a L. donovani strain harbouring a pLEXSY-egfp construct for flow cytometry analysis of the developmental process of promastigote-amastigote differentiation in a host-free system.

Bolhassani et al. integrated pLEXSY-egfp into the genome of L. major and L. infantum and analyzed their promastigote and amastigote stages by fluorescence microscopy and small animal imaging (Figure 1C).


All cited references are available here: www.jenabioscience.com/cms/en/1/browse/1119/


   
    (4) LEXSY products – all you need for cloning, expression, strain selection, and cultivation
   



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