title: Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii.
abstract: Comparing the steady-state expression levels of recombinant proteins in Toxoplasma gondii parasites indicates considerable variability, and this has sometimes caused difficulties in the engineering of transgenic parasites. Anecdotal observations suggested that alteration of the N-terminus, e.g. by engineering as a fusion protein, permits stable expression of various transgenes that were previously difficult to express in their native form. We have exploited the sensitivity and quantitative nature of fire-fly luciferase (LUC) to examine expression levels in further detail. Fusing the 26 N-terminal residues derived from chloramphenicol acetyl transferase (DeltaCAT) to LUC permits efficient transient or stable luciferase expression in transgenic parasite tachyzoites, providing a useful reporter for studies in T. gondii. Site-directed mutagenesis was used to alter the second codon of DeltaCAT-LUC to encode all 20 possible amino acids, and these constructs showed that changes in the second amino acid can have dramatic effects on luciferase activity, with Ala, Glu, and Asp codons yielding the highest expression levels. Similar results were observed for the expression of both GFP and the T. gondii HXGPRT gene, demonstrating the generality of this effect.
authors.affiliation: Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA.
authors.forename: Mariana
authors.initials: M
authors.lastname: Matrajt
authors.forename: Manami
authors.initials: M
authors.lastname: Nishi
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Fraunholz
authors.forename: Oliver
authors.initials: O
authors.lastname: Peter
authors.forename: David S
authors.initials: DS
authors.lastname: Roos
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001482:Base Composition; D015153:Blotting, Western; D003062:Codon / Q000235:genetics; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003062:Codon; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins