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 of 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.

中文翻译：

---

宿主表达系统通过翻译后修饰调节重组 Hsp70 的活性。

使用模式生物进行重组蛋白生产会增加模型特异性的翻译后修饰 (PTM)，这些修饰会影响蛋白质的结构、电荷和功能。70-kDa 热休克蛋白 (Hsp70) 最初被描述为细胞内分子伴侣，具有 ATP 酶和折叠酶活性。最近，已经鉴定了 Hsp70 蛋白（例如，作为免疫调节剂）的新细胞外活性。虽然一些研究表明细胞外 Hsp70 蛋白具有炎症潜力，但其他研究表明具有免疫抑制活性。我们假设在不同表达系统中产生重组 Hsp70 会导致添加不同的 PTM，这或许至少可以解释这些相反的免疫学结果中的一些。大肠杆菌和毕赤酵母，并通过蛋白质制剂的质谱分析，从计算机和体外角度研究 PTM 的影响。DnaK 结构的计算机比较强调存在依赖于表达系统的静电和地形差异。真核系统中 DnaK 的产生极大地影响了其 ATP 酶活性，并显着改变了其下调小鼠树突细胞 (DC) 上 MHC II 和 CD86 表达的能力。DnaK 的磷酸酶处理表明这些差异中的一些与磷酸化特别相关。总之，我们的数据表明 PTM 是表达系统的一个重要特征，其差异会影响 Hsps 与其配体的相互作用以及随后的功能活动。