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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Published on: July 25, 2013
Design of recombinant bacteriocin fusion protein and evaluation of its anticancer and antibacterial activity
Narges Yadollahi1, Taher Mohamadian1, Davoud Esmaeili2
1Department of Microbiology, Shahr-e-Quds Branch, Islamic Azad University, Tehran, Iran.
Background And Objectives:
Gastrointestinal cancer is the fifth most common cause of cancer in the world, and its treatment is currently challenging. Bacteriocins are cationic proteins that are produced by the ribosome. Many bacteriocins exhibit cytotoxicity against cancer cells and are attractive candidates for future research. In this study, the anticancer and antibacterial activity of a recombinant six bacteriocin fusion protein bifidocin, enterocin, nisin, thermophilin, epidermycin, and colicin (BENTEC) was investigated against the AGS cancer cell line and important gram-positive and gram-negative pathogens.
Methods:
The NCBI database provided the gene sequences. The specific ligand of the AGS cell line and the anticancer sequences were added to the recombinant construct. The physical-chemical characteristics of recombinant bacteriocin were assessed by bioinformatics methods. Expression of the recombinant vector pET22b was performed in E. coli BL21(DE3). MTT test was used to evaluate the cytotoxicity in AGS cell lines and normal GES-1 cells. Flow cytometry and Real-Time PCR methods were used to investigate the expression changes of apoptotic genes against the AGS cell line. The anti-bacterial activity of BENTC fusion bacteriocin against Escherichia coli and Enterococcus faecalis was evaluated by MIC, MBC, and disc diffusion methods.
Results:
The recombinant BENTEC fusion bacteriocin contains 424 amino acids, a molecular weight of 47KD, and an isoelectric point of 11.17. The GRAVY score is -0.596, indicating the hydrophilic properties of the fusion bacteriocin. MTT results demonstrated that 52 % of AGS cell lines treated with a concentration of 8 μg/mL of BENTEC fusion bacteriocin were destroyed during 24 hours. Flow cytometry showed that recombinant bacteriocin induced 76.4 % of apoptosis in the treated cell lines (63.7 % early apoptosis and 12.7 % late apoptosis). Real-time RT- PCR indicated that recombinant BENTEC increased expression of the Bax and Caspase 3 genes by 4.53 fold and 5.856 fold, respectively, and decreased gene expression of bcl-2 by 0.18 fold in the AGS line cell. The results demonstrated that the MIC and MBC for E. coli and E. faecalis were 5 μg/ml,10 μg/ml,10 μg/ml, and 20 μg/ml, respectively. Disc diffusion showed that the diameters of the growth inhibition zones for E. coli and E. faecalis were 32 mm and 23 mm, respectively.
Conclusion:
The results of this study showed that the fusion protein of six bacteriocins linked to the anticancer and the specific ligand sequences was able to induce apoptosis in AGS cell line and have antibacterial activity against important gram-positive and gram-negative pathogens at lower concentrations and with greater specificity. Collectively, BENTEC fusion bacteriocin may be regarded as a promising option for anticancer and antibacterial therapy or prophylaxis.
Insights
This study developed a novel fusion protein, BENTEC, demonstrating significant anticancer and antibacterial properties. BENTEC effectively induced apoptosis in gastric cancer cells and inhibited key bacterial pathogens, suggesting its potential for therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Gastrointestinal cancer poses a significant global health challenge.
- Bacteriocins, ribosome-produced cationic proteins, show promise as anticancer agents due to their cytotoxicity.
- Developing novel therapeutic strategies against cancer and resistant pathogens is crucial.
Purpose of the Study:
- To investigate the anticancer and antibacterial activities of a novel recombinant six-bacteriocin fusion protein, BENTEC.
- To evaluate BENTEC's efficacy against the AGS gastric cancer cell line and pathogenic bacteria (E. coli, E. faecalis).
- To explore the molecular mechanisms underlying BENTEC's cytotoxic effects on cancer cells.
Main Methods:
- Gene sequences for six bacteriocins were retrieved and engineered into a recombinant construct with specific ligands.
- Bioinformatic analysis was used to predict the physical-chemical properties of the BENTEC fusion protein.
- Cytotoxicity was assessed using MTT assays, apoptosis was analyzed by flow cytometry, and gene expression (Bax, Caspase 3, bcl-2) was quantified via Real-Time PCR.
- Antibacterial activity was determined using Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and disc diffusion assays.
Main Results:
- The recombinant BENTEC fusion protein (424 amino acids, 47KD) exhibited hydrophilic properties.
- BENTEC demonstrated significant cytotoxicity against AGS cells (52% destruction at 8 µg/mL) and induced 76.4% apoptosis.
- BENTEC upregulated pro-apoptotic genes (Bax, Caspase 3) and downregulated anti-apoptotic gene (bcl-2) in AGS cells.
- Effective antibacterial activity was observed against E. coli and E. faecalis, with MICs of 5-10 µg/mL and MBCs of 10-20 µg/mL.
Conclusions:
- The BENTEC fusion protein effectively induces apoptosis in the AGS gastric cancer cell line.
- BENTEC exhibits potent antibacterial activity against Gram-positive and Gram-negative pathogens.
- The findings suggest BENTEC is a promising candidate for developing novel anticancer and antibacterial therapies.
