Theileria orientalis: cloning a cDNA encoding a protein similar to thiol protease with haemoglobin-binding activity

A gene encoding a protein (Tocp1) from Theileria orientalis was isolated from a cDNA library and the deduced amino acid sequence of Tocp1 has 476 amino acids. The primary structure of Tocp1 is similar to eukaryotic thiol proteases (EC 3.4.22.-), but no enzymatic activity was observed with the substitution of essential cysteine at the cysteine active site for glycine. Southern blot analysis showed that multiple genes similar to Tocp1 were present in the parasite genome. Sequence analysis of the genome of the parasite showed that there are at least five different genes similar to Tocp1. Tocp1 transcripts were detected in the T. orientalis piroplasma by Northern blot analysis. Western blot analysis showed that Tocp1 was expressed in the piroplasm of T. orientalis. To address the role of Tocp1 in the life cycle of T. orientalis, Tocp1 was expressed using pET32 expression system. Binding affinity to haemoglobin was demonstrated by enzyme-linked immunosorbent assay.