Cloning and characterization of selected proteases from the Schistosoma mansoni egg transcriptome.

Daniel Wuthrich

Bachelor's thesis

Abstract:

Schistosomiasis is a prevalent parasitic infection that affects over 210 million individuals worldwide, primarily in regions characterized by inadequate sanitation and limited access to clean water, particularly in tropical and subtropical areas. The disease is caused by the trematodes of the Schistosoma genus, with the eggs being the primary culprits. However, despite their significance, the molecular properties of the proteins encoded in these eggs remain inadequately understood. Shedding light on the egg-host interaction requires a more comprehensive understanding of these proteins, which may aid in developing vaccines against schistosomiasis. This paper focuses on expressing and purifying the following proteases: aminopeptidase-N 1 (APN 1), endothelin-converting enzyme 1 (ECE 1), leishmanolysin-like peptidase (SmLeish), which are the most highly expressed proteases identified in Schistosoma mansoni eggs. We intend to make them accessible for further biochemical studies. To achieve this, we extracted RNA from the eggs and converted it to cDNA, which was then amplified using PCR and ligated into plasmid vectors for transfection into competent cells. Throughout the process, we monitored the presence of the transcripts of interest via sequencing, alignment with BLAST, and gel electrophoresis. After inducing protein expression, we analyzed the proteins using SDS-PAGE. However, we managed to get only SmLeish into the expression stage, but we were getting unsatisfactory results from the SDS-PAGE, preventing us from purifying SmLeish. As for APN 1 and ECE 1, we encountered issues during the ligation into pET-SUMO vector and decided not to repeat the process due to time constraints. Nevertheless, these findings unveiled the difficulties of purifying and working with such proteases, which can provide valuable insights into developing treatments for schistosomiasis.

Keywords

Fluke, Host-parasite interaction, Aminopeptidase-N 1, Endothelin converting enzyme, Leishmanolysin-like peptidase

Language used: English

Date on which the thesis was submitted / produced: 29. 3. 2024

Thesis defence

• Date of defence: 29. 5. 2024
• Supervisor: RNDr. Jan Dvořák, Ph.D.
• Reader: Jiří Černý, Ph.D.

Czech University of Life Sciences Prague

Faculty of Environmental Sciences

Bachelor programme:
Environmental Engineering