Figure 4.
The T. cruzi oligopeptidase B gene is present as a single copy in the genome and expressed in all life cycle stages of the parasite. (A) Southern blot of T. cruzi genomic DNA digests probed with the full-length oligopeptidase B cDNA labeled with [32P]dCTP. The blot was washed to a final stringency of 0.6× SSC at 60°C and exposed to film for 5 d. (B) Expression of the oligopeptidase B gene in life cycle stages of T. cruzi. (I) Amplification of homologous sequences from total parasite RNA by RT-PCR. PCR products were separated on agarose, blotted to nylon, and probed with the fulllength peptidase cDNA, revealing a single band of 2.1 kb in three life cycle stages of T. cruzi. (II) Detection of peptidase activity in parasite soluble extracts separated by SDS-PAGE. Total soluble extract was prepared from each parasite life cycle stage, and 3 × 107 parasite equivalents were loaded in each lane. Oligopeptidase B activity was detected directly in a gel overlay assay as described in the Materials and Methods. Active purified recombinant peptidase (0.6 μg; rPEP) migrated with an apparent molecular mass of 120 kD, similar to oligopeptidase B activity expressed in each developmental stage of T. cruzi: epimastigotes (Epi), metacyclics (Meta), amastigotes (Amast), and trypomastigotes (Trypo).