A new expression vector for Crithidia fasciculata and Leishmania.
Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania. These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers. 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids. We, therefore, inserted the intergenic region of the C. fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene. Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing. A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene. Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein. Biologically active proteins can be expressed in C. fasciculata or L. amazonensis promastigotes and purified by affinity chromatography using a metal chelating column.
Tetaud
Emmanuel
E
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, UK. etetaud@hippocrate.u-bordeaux2.fr
Lecuix
Isabelle
I
Sheldrake
Tara
T
Baltz
Théo
T
Fairlamb
Alan H
AH
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D053804:Granzymes; D049452:Green Fluorescent Proteins; D007891:Leishmania / Q000235:genetics*; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000032:analysis / Q000235:genetics; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C088546:GZMK protein, human; D053804:Granzymes; D012697:Serine Endopeptidases