Trypanosoma bruceirecombinant pyroglutamyl peptidase I, Oligopeptidases A , and B: antigen recognition by infected human sera.
Anywar, Denis Arony
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This work was done to clone, express and purify recombinant oligopeptidases A, B and pyroglutamyl peptidase I from Trypanosoma brucei brucei in order to test the immunoreactivity of these proteins using pooled sera from cases of Human African Trypanosomiasis (HAT). Oligopeptidase A, pyroglutamyl peptidase I (active site present) and pyroglutamyl peptidase I (active site absent) were cloned and expressed in the vectors pQE 30 and pET28a for Oligopeptidase A and only pET28a for both forms of pyroglutamyl peptidase I. Oligopeptidase A gave a protein fragment size of about 65KDa and a smaller one of around 25KDa which were both purified and were non reactive to pooled infected sera from cases of Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Cloning of pyroglutamyl peptidase I (active site absent) was done using site directed mutagenesis involving ligating two separate PCR products that were designed to omit the enzyme active site cysteine (TGC) at position 167 replacing it with (GAG) a Glutamate in the native protein. The two PCR products P1 (amino terminal end) and P2 (carboxyl fragment) were ligated in pGEM T-Easy® cloning vector (Promega) generating two recombinant plasmids pDA-3 and pDA-4. pDA-4 was digested out and ligated into pDA-3 producing pDA-4. pDA-4 was digested and the full length enzyme with the active site cysteine changed to glutamate ligated into pET28a before expression in BL21 DE3 E. coli giving a fragment size of 25.1 KDa. Pyroglutamyl peptidase I (active site present) was cloned by ligating full length sequences (Pfull) in pGEM T-Easy to produce pDA-6 which was then digested out and ligated in pET28a and expressed also in BL21 DE3 E. coli giving a fragment size of 25.1 KDa as well. Pyroglutamyl peptidase I (both forms) reacted weakly with the sensitive luminol detection for both cases of HAT. The expression levels were low below proper detection limits. In both oligopeptidase A and pyroglutamyl peptidases the pooled malaria control sera remained negative. Oligopeptidases B however did not express in both pQE 30 and pET28a expression vectors including in different expression hosts BL21 DE3, MI5, Rosetta® and Rosetta gami cells. This study has demonstrated that PGP I has potential and should further be evaluated as a candidate antigen for HAT diagnosis. Hence optimizations should be carried out to increase expression levels to pave way for analysis using ELISA and other immunological analysis.