Cloning and expression of a cDNA encoding Phosphoribosyl transferase type I from Strongyloides ratti

Strongyloides ratti is closely related to the human parasite S. stercoralis and is commonly used as laboratory model for diagnosis of strongyloidiasis in humans. The enzyme Phosphoribosyl transferase type I (PRTase) of Strongyloides ratti is an important enzyme involved in the salvage of purine nucleotides. RT-PCR amplification of 567 bp cDNA fragment encoding the middle part of a PRTase from S. ratti was carried out using two specific primers. Use of this fragment as a probe allowed the isolation of a larger cDNA sequence through the searching of the expressed sequence tag (EST) database. The entire size of the assembled fragment was 789 bp. The deduced amino acid sequence exhibit a high degree of homology (98.6%) with the only sequence of S. ratt. SrPRT sequence had the lowest genetic distance with the only S. ratti partial mRNA sequence XM_024650090.1 (1.6%). Multiple alignment of SrPRT showed the stretches of amino acid homolog correspond to two putative substrate binding domains for purine and PRPP. In the C-terminus part of the protein, there is also a putative biding domain sequence with high homology. A 642 bp fragment of the SrPRT includes the entire coding sequence corresponding to Met-1 to Lys-214 was expressed into an N-terminal 6His-tag expression PCRT7/NT-TOPO Expression vector in Escherichia coli. A band of 30.5kDa was observed in the IPTG-induced sample compared to the control on SDS-PAGE. Protein expression was confirmed by Western blot analysis using anti-His HRP-conjugated antibody. The successful cloning and expression of PRTase from S. ratti allow us to compare this enzyme with other related proteins. Such knowledge may be valuable for future structure-based drug design strategies using this enzyme as a model system for S. stercoralis.