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Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells
Hanieh Mohammad Pour1, Ali Jahanian-Najafabadi1, Fatemeh Shafiee1
1Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Background:
Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells.
Methods:
In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay.
Results:
The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 μM, respectively.
Conclusion:
In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.
Insights
Researchers developed a novel fusion peptide, TP4-LYC1, combining Tilapia Piscidin 4 and Lycosine-1. This engineered peptide shows potent anti-cancer effects with reduced toxicity to normal cells, offering a safer therapeutic option.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Tilapia Piscidin 4 (TP4) exhibits anti-tumor properties.
- Lycocine-1 (LYC1), an antimicrobial peptide from spider venom, selectively targets cancer cells.
- TP4 demonstrates cytotoxicity to normal cells, necessitating strategies to mitigate this effect.
Purpose of the Study:
- To produce a soluble recombinant fusion peptide (TP4-LYC1) to reduce TP4's cytotoxicity.
- To evaluate the efficacy of the novel chimeric peptide against cancer cells.
Main Methods:
- Optimized induction conditions for expressing TP4-LYC1, TP4, and LYC1 using the pTWIN-1 vector.
- Utilized auto-cleavage of inteins1/2 for peptide purification.
- Assessed cell viability of HeLa and HUVEC cells using MTT assay.
Main Results:
- Optimal conditions for soluble peptide expression involved 0.1 mM IPTG at 22°C.
- Self-cleavage of inteins yielded peptides of 3, 3, and 6 kDa.
- TP4-LYC1 exhibited a lower IC50 (0.83 μM) than TP4 (2.75 μM) against HeLa cells.
Conclusions:
- Successfully produced a novel chimeric peptide, TP4-LYC1.
- The fusion peptide demonstrates potential as a safe biomolecule with potent cytotoxic effects on cancer cells.
- Further studies are needed to determine penetration ability and cell death mechanisms for precise apoptosis induction insights.
