Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis.
For the effective control of African animal trypanosomiasis, there is a great need for the development of point of care diagnostic tests that are affordable to the end-users. Parasite cysteine proteases are involved in the pathogenesis of African trypanosomiasis, hence serve as a good target for the development of new chemotherapeutics and diagnostics. The aim of this study was to evaluate the potential of the cysteine protease TviCATL from Trypanosoma vivax as a chemotherapeutic agent and diagnostic target for African animal trypanosomiasis. The catalytic domain, TviCATL, was recombinantly expressed in Pichia pastoris, auto-catalytically activated by low pH removal of the proregion, purified by three phase partitioning and molecular exclusion chromatography to homogeneity and its identity confirmed by western blot analysis. Endoglycosidase H treatment of TviCATL indicated that the protease is Asn-glycosylated. The pH optimum was determined to range between 6.5 and 7.5 suggesting that the enzyme would be active in the host bloodstream following parasite lysis. The protease was able to degrade the host proteins: bovine serum albumin, bovine haemoglobin, gelatin, type I collagen and the endogenous protease inhibitor, bovine α2-macroglobulin in vitro at neutral pH. The peptidolytic specificity of the protease was determined by considering the active site binding pocket S2- substrate -P2 interaction. TviCATL showed high endopeptidase specificity for Z-Phe-Arg-AMC and H-D-Ala-Leu-Lys-AMC, suggesting that the hydrophobic residues Phe or Leu are favoured at the P2 position in the presence of basic Arg or Lys at P1 position. The TviCATL peptidolytic activity was inhibited by E-64, iodoacetate, leupeptin, antipain, Z-Gly-Leu-Phe-CMK (albeit at a reduced level) and iodoacetamide inhibitors and this indicated that TviCATL is a cysteine protease. Satisfactory anti-TviCATL antibody levels were produced in chickens as evidenced by good signals in the ELISA and western blot analysis. The specificity and affinity of chicken anti-TviCATL IgY antibodies for TviCATL antigen was improved by affinity purification of these antibodies using a TviCATL-affinity column. The serodiagnostic potential of the TviCATL antigen and cross-reactivity with anti-T. congolense antibodies (in sera) was evaluated in an antibody inhibition ELISA format. These antibodies were able to discriminate between non-infected cattle sera and T. congolense-infected sera, thus suggesting some degree of cross-reactivity between TviCATL antigen and anti-T. congolense antibodies. As an alternative to animal-based antibody production, the Nkuku® phage library was used to select for single-chain variable fragment (scFvs) antibodies by panning against TviCATL antigen. As evaluated by polyclonal and monospecific phage enzyme-linked immunosorbent assay (ELISA), after the third round of panning, the TviCATL-scFvs binders were enriched. However, only one clone gave a promising ELISA signal suggesting that further optimisation of panning conditions are required in order to obtain improved monospecific ELISA signals for application of TviCATL-scFvs in diagnostic assays. TviCATL therefore shows promise as both a chemotherapeutic and diagnostic target to African animal trypanosomiasis.