Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537815v1?rss=1 Authors: Paterou, A., Tyc, J., Sunter, J., Vaughan, S., Gull, K., Dean, S. Abstract: African trypanosomes are medically important parasites that cause Sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of greater than 100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC