Background: Heat shock proteins (HSPs) represent a group of important proteins which are produced by all kinds of organisms especially under stressful conditions. DnaK, an Hsp70 homolog in prokaryotes, has indispensable roles when microbes was confronted with stress conditions. However, few data on DnaK from Rhodococcus sp. were available in the literature. In a previous study, we reported that toluene and phenol stress gave rise to a 29.87-fold and 3.93-fold increase for the expression of DnaK from R. ruber SD3, respectively. Thus, we deduced DnaK was in correlation with the organic solvent tolerance of R. ruber SD3.

Objective: To elucidate the role of DnaK in the organic solvent tolerance of R. ruber SD3, expression, purification and functional analysis of Dnak from R. ruber SD3 were performed in the present paper.

Methods: In this article, DnaK from R. ruber SD3 was heterologously expressed in E. coli BL21(DE3) and purified by affinity chromatography. Functional analysis of DnaK was performed using determination of kinetics, docking, assay of chaperone activity and microbial growth.

Results: The recombinant DnaK was rapidly purified by affinity chromatography with the purification fold of 1.9 and the recovery rate of 57.9%. Km, Vmax and Kcat for Dnak from R. ruber SD3 were 80.8 μM, 58.1 nmol/min and 374.3 S^-1, respectively. The recombinant protein formed trimer in vitro, with the calculated molecular weight of 214 kDa. According to in-silico analysis, DnaK interacted with other molecular chaperones and some important proteins in the metabolism. The specific activity of catalase in the presence of recombinant DnaK was 1.85 times or 2.00 times that in the presence of BSA or Tris-HCl buffer after exposure to 54 °C for 1h. E. coli transformant with pET28-dnak showed higher growth than E. coli transformant with pET28 at 43°C and in the presence of phenol, respectively.

Conclusion: The biochemical properties and the interaction analysis of DnaK from R. ruber SD3 deepened our understanding of DnaK function. DnaK played an important role in microbial growth when R. ruber was subjected to various stress such as heating and organic solvent.

背景：热休克蛋白（HSPs）代表了一组重要的蛋白质，由各种生物特别是在压力条件下产生。DnaK 是原核生物中的 Hsp70 同源物，当微生物面临压力条件时，它具有不可或缺的作用。然而，很少有关于来自红球菌属的 DnaK 的数据。可在文献中找到。在之前的一项研究中，我们报道了甲苯和苯酚压力分别使来自红红树莓 SD3 的 DnaK 的表达增加了 29.87 倍和 3.93 倍。因此，我们推断 DnaK 与 R. ruber SD3 的有机溶剂耐受性相关。

目的：为了阐明DnaK在R. ruber SD3有机溶剂耐受性中的作用，本文对R. ruber SD3中Dnak的表达、纯化和功能进行了分析。

方法：在这篇文章中，来自红色红血球 SD3 的 DnaK 在大肠杆菌 BL21(DE3) 中异源表达，并通过亲和层析纯化。使用动力学测定、对接、分子伴侣活性测定和微生物生长来进行 DnaK 的功能分析。

结果：重组DnaK经亲和层析快速纯化，纯化倍数为1.9，回收率为57.9%。R. ruber SD3 的 Dnak 的 Km、Vmax 和 Kcat 分别为 80.8 μM、58.1 nmol/min 和 374.3 S ^-1。重组蛋白在体外形成三聚体，计算分子量为214 kDa。根据计算机分析，DnaK 与其他分子伴侣和代谢中的一些重要蛋白质相互作用。在重组 DnaK 存在下过氧化氢酶的比活性是在 BSA 或 Tris-HCl 缓冲液中暴露于 54°C 1 小时后的 1.85 倍或 2.00 倍。分别在 43°C 和苯酚存在下，带有 pET28-dnak 的大肠杆菌转化体的生长速度高于带有 pET28 的大肠杆菌转化体。

结论：R. ruber SD3中DnaK的生化特性和相互作用分析加深了我们对DnaK功能的理解。当R. ruber受到加热和有机溶剂等各种压力时，DnaK在微生物生长中发挥了重要作用。