Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii

Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA.
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 05/2002; 120(2):285-9. DOI: 10.1016/S0166-6851(02)00014-2
Source: PubMed


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.

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    • "The parasite’s virulence and ability to cause systemic infection can be visualized in real time using parasites engineered to express luciferase and hosts injected with the enzyme’s substrate, luciferin [50,51]. After interperitoneal injection, luciferin distributes quickly and without regard for any blood-tissue barrier [52]. "
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    • "Comparison with the consensus sequence from T. gondii translational start sites (Seeber 1997) supports the notion that the EtMIC4 start codon is in a favourable context (e.g. A −1 , A −3 and G −6 ), although further downstream homology is less obvious (Matrajt et al. 2002). RT-PCR and EST data published since the original EtMIC4 prediction now show that the sequence encoding the MGFFVFTG previously proposed to be the start methionine and part of the signal peptide (Tomley et al. 2001) is in fact partially located within the third intron (supplementary Fig. S2). "
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    • "In order to construct a renilla version of the DCAT-pluc-firefly luciferase (Matrajt et al., 2002), the renilla coding region was amplified by PCR with primers that incorporated Avr-II/Pst-1 restriction sites. This DNA fragment was then used to replace the firefly coding region in the DCAT-pluc-firefly luciferase plasmid. "
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