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  • Title: Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae.
    Author: Kushnir S, Gase K, Breitling R, Alexandrov K.
    Journal: Protein Expr Purif; 2005 Jul; 42(1):37-46. PubMed ID: 15939291.
    Abstract:
    Production of functional eukaryotic proteins in recombinant form is a bottle-neck in various post-genomic applications and in life science in general. At least partially this is due to the problems associated with the use of endogenous RNA polymerase II for high-level transcription of heterologous genes in eukaryotic expression systems. To circumvent these problems we developed a new inducible protein expression system based on the protozoan host Leishmania tarentolae (Trypanosomatidae). We have created a strain of L. tarentolae constitutively co-expressing T7 RNA polymerase and tetracycline repressor. This strain could be stably transformed with the heterologous target gene under control of the T7 promoter/TET operator assembly, which can initiate transcription upon addition of tetracycline to the culture medium. Using this system, we demonstrated that enhanced green fluorescent protein (EGFP) could be overexpressed to a level of ca. 1% of total cellular protein. The developed system was tested for its ability to inducibly co-express multiple genes. Using two copies of the egfp gene integrated at two different genomic sites, we could obtain expression levels reaching 4% of total cellular protein. Further possible improvements and applications of the developed system are discussed.
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