Title

Purification of a Recombinant Paraflagellar Rod Protein-5 from Trypanosoma cruzi

Document Type

Oral Presentation

Campus where you would like to present

Ellensburg

Event Website

https://digitalcommons.cwu.edu/source

Start Date

16-5-2021

End Date

22-5-2021

Keywords

Protein Purification, Trypanosoma cruzi, Paraflagellar rod

Abstract

Trypanosoma cruzi is a single-celled parasite found in the Americas, from the southern United States through South America, and is responsible for causing Chagas disease. This vector-borne parasite can infect all mammals, including humans, through exposure to the feces of Kissing bugs (Triatomine bugs). These large bugs feed on blood and can infest mud-walls or thatched roofs of rural homes. Trypanosomes contain a distinct structure known as the paraflagellar rod (PFR) that runs alongside the parasite’s flagellum. The PFR is required for cell motility and is a complex structure of cytoskeletal filaments in a lattice-like arrangement. The PFR constitutes an immunologically unique set of proteins that have been shown to protect against T. cruzi infection in mice. Paraflagellar rod protein-5 (PFR-5) is a putative minor component of the PFR of Trypanosomes that contains both PFR and SH3 protein domains. This research aims to identify the sub-cellular location of PFR-5 within the parasite T. cruzi. The 5’ end of the pfr5 gene has been subcloned into the PinPoint Xa expression plasmid in Escherichia coli (JM109) cells. My research has focused on induction, extraction, and purification of the soluble PFR-5 fragment. Protein purification is completed with pH manipulation of strep-avidin beads for protein collection and verified through SDS-PAGE and western blot. The goal is to immunize mice with the collected protein to generate PFR-5 specific antibodies for fluorescent microscopy. This research contributes to the understanding of the PFR in Trypanosomes and increases the number of potential vaccine target proteins for Chagas disease.

Faculty Mentor(s)

Gabrielle Stryker

Department/Program

Biological Sciences

Additional Mentoring Department

https://cwu.studentopportunitycenter.com/purification-of-a-recombinant-paraflagellar-rod-protein-5-from-trypanosoma-cruzi/

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May 16th, 12:00 PM May 22nd, 12:00 PM

Purification of a Recombinant Paraflagellar Rod Protein-5 from Trypanosoma cruzi

Ellensburg

Trypanosoma cruzi is a single-celled parasite found in the Americas, from the southern United States through South America, and is responsible for causing Chagas disease. This vector-borne parasite can infect all mammals, including humans, through exposure to the feces of Kissing bugs (Triatomine bugs). These large bugs feed on blood and can infest mud-walls or thatched roofs of rural homes. Trypanosomes contain a distinct structure known as the paraflagellar rod (PFR) that runs alongside the parasite’s flagellum. The PFR is required for cell motility and is a complex structure of cytoskeletal filaments in a lattice-like arrangement. The PFR constitutes an immunologically unique set of proteins that have been shown to protect against T. cruzi infection in mice. Paraflagellar rod protein-5 (PFR-5) is a putative minor component of the PFR of Trypanosomes that contains both PFR and SH3 protein domains. This research aims to identify the sub-cellular location of PFR-5 within the parasite T. cruzi. The 5’ end of the pfr5 gene has been subcloned into the PinPoint Xa expression plasmid in Escherichia coli (JM109) cells. My research has focused on induction, extraction, and purification of the soluble PFR-5 fragment. Protein purification is completed with pH manipulation of strep-avidin beads for protein collection and verified through SDS-PAGE and western blot. The goal is to immunize mice with the collected protein to generate PFR-5 specific antibodies for fluorescent microscopy. This research contributes to the understanding of the PFR in Trypanosomes and increases the number of potential vaccine target proteins for Chagas disease.

https://digitalcommons.cwu.edu/source/2021/COTS/16