title: Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae.
abstract: 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.
authors.affiliation: Department of Chemical Biology Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Susanna
authors.initials: S
authors.lastname: Kushnir
authors.forename: Ion Cristian
authors.initials: IC
authors.lastname: Cirstea
authors.forename: Lyudmyla
authors.initials: L
authors.lastname: Basiliya
authors.forename: Nataliya
authors.initials: N
authors.lastname: Lupilova
authors.forename: Reinhard
authors.initials: R
authors.lastname: Breitling
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques; D022201:Chromosomes, Artificial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D007891:Leishmania / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases