Therapeutic peptides for Chagas disease: a new approach to cardiovascular diseases
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Chagas disease affects approximately 8 million people globally, primarily in South and Central America, but also in several European countries, Japan, Australia, the United States and Canada.Más información
Chagas disease affects approximately 8 million people globally, primarily in South and Central America, but also in several European countries, Japan, Australia, the United States and Canada. Chagas has become a global health threat with an economic burden of USD7.19 billion annually due to healthcare costs associated with Chagas cardiovascular diseases. Chagas’ acute stage starts with flu-like symptoms and can develop into a chronic stage that is often manifested by cardiac or gastrointestinal diseases. Chronic cardiac Chagas symptoms include enlarged heart, arrhythmia, stroke, and death. New drugs are urgently needed because those that are currently available are highly toxic and ineffective in treating the chronic stage. Challenges in developing drugs for Chagas disease include the lack of effective drug candidates and limited knowledge of drug targets and mechanisms of action. This project aims to develop new protein inhibitor candidates against T. cruzi, the protozoan parasite that causes Chagas.
This project was selected for funding during the first research competition of the Joint Canada-Israel Health Research Program – Phase II. The program is a partnership between IDRC, the Canadian Institutes of Health Research, the Israel Science Foundation and the Azrieli Foundation.
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Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 du/ µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 du/ µg plasmid DNA. The protocol generates sufficient transformants for 10-100 x coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method' s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for down stream screening purposes while limiting the amount of the genomic library required.
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