BACKGROUND: Molecular chaperones regulate the proper folding of proteins in the cell. Members of the Hsp70 family, including the Ssa1 protein, are molecular chaperones that prevent protein aggregation, promote their proper folding and degradation, and are the most common among the various chaperones, highly conserved, and present in a variety of organisms. AIM: The aim of the work was to optimize methods for the production, extraction and purification of Ssa1 protein from cells of Saccharomyces cerevisiae. MATERIALS AND METHODS: The SSA1-4 gene sequences were cloned into a vector under the control of the TEF1 promoter and fused with a sequence encoding His6-tag. Yeast strains with different genetic backgrounds were transformed with the obtained constructs, and the production of Ssa1-4 proteins was assessed under different cultivation conditions. Affinity and ion-exchange chromatography were used to purify the Ssa1 protein. Fluorescence microscopy was used to confirm the localization of recombinant Ssa proteins fused with TagRFP-T in the cytosol. RESULTS AND CONCLUSIONS: Methods for the production, extraction and purification of Ssa1 protein from yeast cells have been optimized. The same approach can be further used to purify other Hsp70 proteins and adapted to obtain various proteins from eukaryotic cells.