Article

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: 2.55). 05/2002; 120(2):285-9. pp.285-9
Source: PubMed

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.

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Keywords

20 possible amino acids
 
26 N-terminal residues
 
Anecdotal observations
 
Asp codons
 
chloramphenicol acetyl transferase
 
fire-fly luciferase
 
fusion protein
 
luciferase activity
 
permits stable expression
 
recombinant proteins
 
second amino acid
 
second codon
 
Site-directed mutagenesis
 
stable luciferase expression
 
T. gondii
 
T. gondii HXGPRT gene
 
Toxoplasma gondii parasites
 
transgenic parasite tachyzoites
 
transgenic parasites
 
various transgenes