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Updated: Jul 24, 2026
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
Gene cloning and characterization of a novel recombinant 40-kDa heat shock protein from Mesobacillus persicus B48
Fatemeh Fathinejad1, Hossein Ghafouri2,3, Ebrahim Barzegari4
1Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.
Abstract:
The present study reports the recognition and characterization of the gene encoding the co-chaperone DnaJ in the halophilic strain Mesobacillus persicus B48. The new extracted gene was sequenced and cloned in E. coli, followed by protein purification using a C-terminal His-tag. The stability and function of the recombinant DnaJ protein under salt and pH stress conditions were evaluated. SDS-PAGE revealed a band on nearly 40-kDa region. The homology model structure of new DnaJ demonstrated 56% similarity to the same protein from Streptococcus pneumonia. Fluorescence spectra indicated several hydrophobic residues located on the protein surface, which is consistent with the misfolded polypeptide recognition function of DnaJ. Spectroscopic results showed 56% higher carbonic anhydrase activity in the presence of the recombinant DnaJ homolog compared to its absence. In addition, salt resistance experiments showed that the survival of recombinant E. coli+DnaJ was 2.1 times more than control cells in 0.5 M NaCl. Furthermore, the number of recombinant E. coli BL21+DnaJ colonies was 7.7 times that of the control colonies in pH 8.5. Based on the results, DnaJ from the M. persicus can potentially be employed for improving the functional features of enzymes and other proteins in various applications.
Insights
Researchers identified and characterized the DnaJ co-chaperone from Mesobacillus persicus B48. This halophilic DnaJ protein enhances enzyme activity and improves bacterial salt and pH tolerance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Chaperones like DnaJ are crucial for protein folding and stability.
- Halophilic bacteria possess unique proteins adapted to extreme environments.
- Understanding DnaJ function in halophiles can reveal novel biotechnological applications.
Purpose of the Study:
- To identify and characterize the DnaJ co-chaperone gene from the halophilic bacterium Mesobacillus persicus B48.
- To evaluate the stability and functional properties of the recombinant M. persicus DnaJ protein.
- To explore the potential applications of M. persicus DnaJ in enhancing protein stability and function.
Main Methods:
- Gene cloning and expression in E. coli.
- Protein purification using C-terminal His-tag.
- SDS-PAGE for molecular weight determination.
- Homology modeling for structural analysis.
- Fluorescence spectroscopy to assess protein surface properties.
- Enzyme activity assays (carbonic anhydrase).
- Salt and pH stress resistance experiments in E. coli.
Main Results:
- The DnaJ gene from M. persicus was successfully cloned, expressed, and purified.
- The recombinant DnaJ protein showed stability under salt and pH stress.
- Homology modeling indicated 56% similarity to Streptococcus pneumoniae DnaJ, with surface hydrophobic residues.
- Enzyme activity of carbonic anhydrase increased by 56% in the presence of recombinant DnaJ.
- Recombinant E. coli expressing DnaJ showed 2.1-fold increased survival in 0.5 M NaCl and 7.7-fold higher colony count at pH 8.5 compared to controls.
Conclusions:
- The DnaJ co-chaperone from M. persicus is a functional protein with enhanced stability under stress conditions.
- M. persicus DnaJ can significantly improve the activity and stability of other proteins, such as carbonic anhydrase.
- This halophilic DnaJ has potential applications in biotechnology for enhancing enzyme performance and microbial stress tolerance.
