Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae

• Published in: Molecular and biochemical parasitology Vol. 176; no. 2; pp. 69 - 79
• Main Authors: Kushnir, Susanna, Cirstea, Ion Cristian, Basiliya, Lyudmyla, Lupilova, Nataliya, Breitling, Reinhard, Alexandrov, Kirill
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-04-2011; Amsterdam: Elsevier
• Subjects: Blotting, Western; Cell Culture Techniques; Chromosomes, Artificial; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Electrophoresis, Polyacrylamide Gel; Electroporation; Episomal elements; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Engineering - methods; Genetic Vectors - metabolism; Genome; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Kinetoplastida; Leishmania; Leishmania - genetics; Leishmania - metabolism; Leishmania tarentolae; Plasmids - genetics; Plasmids - metabolism; Protein Processing, Post-Translational; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant protein expression; Transfection - methods; Kinetoplastida; Gene expression; Leishmania; Episomal elements; Recombinant protein expression
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2010.12.002

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▶ The protozoon
Leishmania tarentolae was developed as a host for recombinant protein production. ▶ We present a new type of expression architecture for
L. tarentolae based on linear elements. ▶ Overexpression of recombinant proteins in transgenic organisms reaches up to 30% of total cellular protein using this architecture.
The trypanosomatid protozoon
Leishmania tarentolae is a well-established model organism for studying causative agents of several tropical diseases that was more recently developed as a host for recombinant protein production. Although several expression architectures based on foreign RNA polymerases have been established for this organism, all of them rely on integration of the expression cassette into the genome. Here, we exploit a new type of expression architecture based on linear elements. These expression vectors were propagated in
Escherichia coli as circular plasmids and converted into linear episomes with telomere-like structures prior to transfection of
L. tarentolae. Overexpression of recombinant proteins in transgenic organisms exceeding 10% of total cellular protein, one of the highest overexpression levels obtained in a eukaryotic organism for a cytosolic protein. We show that the linear elements are stably propagated in
L. tarentolae cells over long periods of time (>90 generations) without major changes in structure or expression yields. Overexpressing cultures can be obtained without clonal selection of the transfected cells. To establish the utility of the developed system for protein production in a parallelized format, we expressed 37 cytosolic, peripheral, and membrane proteins as fusions with EGFP in
L. tarentolae using linear vectors. We detected the expression of 30 of these targets and describe the preparative purification of two arbitrarily selected proteins.