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Vacuolar aspartic protease of Candida albicans
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Aspartic proteases (APs) are crucial for diverse cellular processes. This thesis delves into the complexities of protein expression and characterization of vacuolar aspartic endoprotease Apr1p from Candida albicans, comparing it to its Saccharomyces cerevisiae ortholog, Pep4p. Recombinant expression of Apr1p in Escherichia coli yielded the inactive proenzyme, proApr1p. Extensive refolding efforts failed to produce mature, active Apr1p, suggesting a reliance on intricate cellular machinery or specific post-translational modifications for activation. Attempts to leverage vacuolar enzymes or cell lysates for proApr1p activation were unsuccessful, potentially due to the fragility of isolated vacuoles and the complex mixture of enzymes in cell lysates. Positive results emerged when Apr1p was expressed in S. cerevisiae, where fractionated cell lysates exhibited specific proteolytic activity at acidic pH after inhibiting serine and metalloproteases proteases. The eukaryotic system can probably produce active Apr1p. However, after preliminary small-scale experiments, upscaling of Apr1p expression in S. cerevisiae will be necessary in order to obtain sufficient amount of protein for further characterization. A reciprocal gene swap experiment, exchanging PEP4 in S. cerevisiae with APR1 and vice versa,...
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