The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.,This article is protected by copyright. All rights reserved.