Molecular cloning of the Toxoplasma gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein

Abstract

An 18 kDa bradyzoite specific surface protein of Toxoplasma gondii (T. gondii) has been purified by affinity chromatography with a specific monoclonal antibody using parasites grown in vitro under conditions inducing the biosynthesis of bradyzoite specific proteins. N-terminal and internal amino acid sequences obtained by microsequencing enabled us to design degenerate oligonucleotides. A fragment of 187 bp was amplified by polymerase chain reaction (PCR). It was used as a probe to clone a 4 kb-Bam HI fragment encompassing the gene encoding the 18 kDa protein. Nucleotide sequence analysis revealed a single open reading frame of 516 nucleotides encoding a 172 amino acid protein. The deduced amino acid sequence matched perfectly the peptides microsequenced from the native protein. The N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 27 amino acids. The hydrophobic C-terminal part could represent a signal for a glycan-phosphoinositide anchor. The full-length cDNA was also isolated and both the 5' and 3' untranslated regions were determined. Reverse transcriptase-PCR (RT-PCR) using p18-specific primers showed a stage-specific expression of this gene. Comparison of the nucleic acid sequence and the predicted amino acid sequence with databases did not reveal significant homology with known genes or proteins. This gene is proposed to be named sag4, according to the existing T. gondii nomenclature.