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Molecular chaperones are highly conserved, ubiquitous enzymes that maintain proteostasis by mediating protein structure in vivo. Post-translational modifications (PTMs) can act as a functional switch between chaperone pathways, allowing for cells to enact a coordinated response to cellular perturbations. Several PTMs are well characterized in the context of chaperone function, though the role of N-terminal acetylation is commonly overlooked, leading to an overwhelming lack of scientific understanding in this area of study. Ssa1, a Hsp70 class chaperone endogenous to Saccharomyces cerevisiae, is likely N-terminally acetylated, though in vitro studies of Ssa1 are limited by current purification methods which are resource-intensive. To address these limitations, we developed a one-step purification scheme that uses a Protein-A fusion transformant strain in combination with IgG-conjugated magnetic Dynabeads to facilitate Ssa1 pull-down. Using this method, Ssa1 was natively isolated in high purity and yield while retaining its activity and our modification of interest. This study also established the basis for evaluating differences Ssa1 chaperone activity due to disruption of N-terminal acetylation by purifying Ssa1 in N-terminal acetylation deficient backgrounds. Through the expression and purification of the Ssa1 co-chaperone, Ydj1, this study has additionally provided components needed to facilitate future co-chaperone binding experiments, which will help determine the contribution of N-terminal acetylation to Hsp70 chaperone activity and efficiency.

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