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Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein
Meysam Soleimani1, Hamid Mirmohammad-Sadeghi1, Hojjat Sadeghi-Aliabadi2
1Department of Pharmaceutical Biotechnology, Isfahan University of Medical Sciences, Isfahan, Iran.
Background:
Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein.
Materials And Methods:
In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains.
Results:
Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins.
Conclusions:
The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains.
Insights
Researchers optimized the expression of a novel p28-NRC chimeric protein for cancer therapy. Using thioredoxin (Trx) and 6x-His tags, they achieved successful cloning, expression, and purification of this targeted therapeutic agent.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Therapeutics
Background:
- Chimeric proteins combining targeting and cytotoxic moieties are key in cancer targeted therapy.
- This study focuses on the p28-NRC chimeric protein, using p28 peptide for targeting/anticancer effects and NRC peptide for cytotoxicity.
- The antimicrobial activity of NRC peptide presents challenges for expression in Escherichia coli.
Purpose of the Study:
- To clone, express, and purify the p28-NRC chimeric protein.
- To evaluate the impact of thioredoxin (Trx) and 6x-His fusion tags on expression.
- To optimize expression conditions and host strains for improved yield.
Main Methods:
- Utilized pET28 and pET32 expression plasmids for the chimeric protein with 6x-His and both Trx and 6x-His tags, respectively.
- Optimized expression conditions and host strains.
- Evaluated expression using SDS-PAGE and Western blot analysis.
Main Results:
- Low expression yield was observed with the pET28 plasmid (6x-His tag only).
- Optimization of conditions and host strains in the pET32 vector (Trx and 6x-His tags) resulted in reasonable expression yield.
- A molecular weight deviation of approximately 10 kDa was noted in SDS-PAGE for purified proteins from both plasmids.
Conclusions:
- Proper expression and purification yield of the p28-NRC chimeric protein were achieved using the Trx tag.
- Optimization of culture conditions and host strains is crucial for expressing toxic chimeric proteins.
