Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae.
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.
Kushnir
Susanna
S
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
Gase
Klaus
K
Breitling
Reinhard
R
Alexandrov
Kirill
K
D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D007891:Leishmania / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; C034811:tetracycline resistance-encoding transposon repressor protein; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D013752:Tetracycline