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Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach
Mortaza Taheri-Anganeh1, Navid Nezafat2,3, Saba Gharibi4
1Cellular and Molecular Research Center, Cellular and Molecular Medicine Research Institute, Urmia University of Medical Sciences, Urmia, Iran.
Background:
Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form.
Methods:
Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures.
Results:
According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures.
Conclusion:
The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.
Insights
Researchers identified ideal signal peptides for producing secretory and soluble RTX-A, a pore-forming toxin, in Bacillus subtilis. This discovery advances novel cancer treatment strategies by improving protein production and purification for potential therapeutic applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Cancer remains a leading global cause of death, necessitating novel therapeutic strategies beyond conventional treatments.
- Sea anemone actinoporins, like RTX-A, are pore-forming toxins (PFTs) with membranolytic properties, showing potential for cancer therapy.
- Producing recombinant proteins in a secretory form offers advantages in solubility and purification for therapeutic development.
Purpose of the Study:
- To identify optimal signal peptides for the secretory production of RTX-A in Bacillus subtilis.
- To ensure the produced RTX-A is soluble, stable, and non-allergenic for potential therapeutic use.
Main Methods:
- Bioinformatic tools, including Signal Peptide Web Server, SignalP, ProtParam, Protein-sol, and AlgPred, were employed for signal peptide selection and evaluation.
- Physico-chemical properties, solubility, allergenicity, and structural integrity (secondary and tertiary) of RTX-A fused with signal peptides were predicted.
- Structure validation was performed using the PROCHECK server.
Main Results:
- Bioinformatic analysis identified OSMY_ECOLI and MALE_ECOLI as suitable signal peptides for fusion with RTX-A.
- The resulting fusion proteins were predicted to be stable, soluble, and non-allergenic.
- Predicted secondary and tertiary structures of the fusion proteins were deemed appropriate.
Conclusions:
- The identified signal peptides are promising for the rational design of secretory and soluble RTX-A production.
- Further experimental validation and patent exploration of these signal peptides are recommended for advancing RTX-A-based cancer therapeutics.
