Development of high-yield in vitro and in vivo protein expression systems based on Leishmania tarentolae

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Generation of recombinant proteins is a key methodology for Life Science in general and the biotechnological industry in particular. Despite over 30 year long history production of eukaryotic proteins in recombinant form remains problematic. To a significant extent this is due to the fact tfiat all available eukaryotic protein expression Systems utilize the endogenous and highiy regulated RNA polymerase II for heterologous gene transcription. The complexity of the transcription control machinery precludes engineering eukaryotes ttiat reproducibly produce large amounts of recombinant polypeptides. In order to develop a reliable and high-yield eukaryotic expression System we exploited the fast growing non-pathogenic Kinetoplastida Leishmania tarentolae. The feature that sets Kinetoplastida apart form other eukaryotes is the ability to translate messenger RNA generated by RNA polymerase I or even a foreign polymerase due to natural uncoupling of transcription from RNA processing. In the most successfui implementation the heterologous gene is delivered on a circular episome into the L.tarentoale strain. Although this System was shown to deliver up to 300mg of recombinant protein per liter it suffers from strong clonal variations. Initial results suggests that this may be linked to the behavior of episomal elements in Leishmania cells. Understanding of structural dynamics, inheritance and transcription of the circular and linear episomes information should be essential for constructing new generations of expression vectors and for developing strategies in suppressing episome carrying drug resistance genes in pathogenic strains. In a complementary approach, we plan to develop a high-yield in vitro translation System based on extracts of Ltarentolae. Such a System is expected to be more robust and much less expensive tJian other in vitro translation Systems.