Expression, Rapid Purification and Functional Analysis of DnaK from Rhodococcus ruber

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Heat shock proteins (HSPs) are crucial for cellular response to stress.
• DnaK, an Hsp70 homolog in prokaryotes, plays vital roles under stress conditions.
• Limited data existed on DnaK from Rhodococcus sp., particularly its role in organic solvent tolerance.

Purpose of the Study:

• To investigate the function of DnaK in Rhodococcus ruber SD3 concerning organic solvent tolerance.
• To express, purify, and functionally analyze DnaK from R. ruber SD3.

Main Methods:

• Heterologous expression of DnaK in E. coli BL21(DE3).
• Purification of recombinant DnaK using affinity chromatography.
• Functional analysis including kinetic determination, docking, chaperone activity assays, and microbial growth studies.

Main Results:

• Recombinant DnaK was purified with a 1.9-fold purification and 57.9% recovery.
• Kinetic parameters (Km, Vmax, Kcat) were determined for R. ruber SD3 DnaK.
• In silico analysis suggested DnaK interacts with other chaperones and metabolic proteins.
• DnaK enhanced catalase activity at high temperatures and improved E. coli growth under heat and phenol stress.

Conclusions:

• Biochemical characterization and interaction analysis of R. ruber SD3 DnaK provide insights into its function.
• DnaK is important for microbial growth under various stress conditions, including heat and organic solvents.