Cloning and Expression of a cDNA Encoding Phosphoribosyltransferase Type I From Strongyloides ratti

Introduction: Strongyloides ratti is closely related to the human parasite Strongyloides stercoralis and is commonly used as a laboratory model for studying and diagnosing strongyloidiasis in humans. The enzyme phosphoribosyltransferase (PRTase) type I of S. ratti is an important enzyme involved in the salvage of purine nucleotides. 
Materials & Methods: Reverse transcription-polymerase chain reaction (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. The use of this fragment as a probe allowed the isolation of a larger cDNA sequence through searching the expressed sequence tag (EST) database.
Results: The entire size of the assembled fragment was 789 bp. The deduced amino acid sequence exhibits a high degree of homology (98.6%) with the only sequence of S. ratti. The S. ratti phosphoribosyltransferase (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 that the stretches of amino acid homology correspond to two putative substrate-binding domains for purine and phosphoribosyl diphosphate (PRPP). In the C-terminus part of the protein, there is also a putative binding domain sequence with high homology. A 642 bp fragment of the SrPRT, including the entire coding sequence corresponding to Met-1 to Lys-214 was expressed into an N-terminal 6His-tagPCRT7/NT-TOPO expression vector in Escherichia coli. A band of 30.5 kDa was observed in the IPTG-induced sample compared to the control on SDS-PAGE. Protein expression was confirmed by Western blot analysis using an anti-His HRP-conjugated antibody. 
Conclusion: 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.