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
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.