Protein Engineering and Precise Modulation of Protein Expression in the Protist Parasite Trypanosoma Brucei

The unicellular parasite Trypanosoma brucei is the causative agent of African sleeping sickness in humans and Nagana in livestock, diseases that result in significant health and economic burden in Sub-Saharan Africa. The organism’s unusual strategy for evading the immune system is based on its unique surface coat, which consists of a dense, repetitive array of a single Variant Surface Glycoprotein (VSG). We have employed multiple engineering-based approaches to alter the makeup of this VSG coat in order to examine interactions between the parasite and its host’s immune response. Although our work has largely focused on immunology and parasitology, the technology we have developed should be broadly applicable to many systems, and may therefore be of interest to the synthetic biology community. Here, we demonstrate a robust enzymatic method for covalently attaching diverse molecules to engineered surface VSGs of live parasites. We also present a novel technique for modulating protein expression. Parasites were engineered to stably express a second surface VSG with varied expression levels ranging nearly two orders of magnitude. Modulation in protein expression was achieved by varying the genomic insertion point of the second VSG gene, and by altering the 3’ and 5’ untranslated regions flanking the gene.