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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy
Ângela Sousa1, João A Queiroz, Fani Sousa
1CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal.
Abstract:
The gene therapy approach based on reestablishment of p53 tumor suppressor, which acts as a prevailing guardian against malignant cell transformation, is raising new prospects on the outcome of an effective anticancer treatment. It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Therefore, several downstream methods have been proposed to achieve high quantities of supercoiled plasmid DNA with pharmaceutical grade purity. Affinity chromatography with amino acids as ligands has recently yielded interesting results because these ligands take advantage of their biological function or chemical structure to promote specific interactions with different nucleic acids. Here, we describe detailed procedures for the preparation and purification of supercoiled plasmid DNA, with the purity degree required by regulatory agencies, by using arginine affinity chromatography. With this methodology pure pDNA is obtained, efficient on eukaryotic cell transfection and biologically active, resulting in the reestablishment of the p53 protein levels in cancer cell lines.
Insights
Gene therapy using p53 tumor suppressor shows promise for cancer treatment. Arginine affinity chromatography effectively purifies plasmid DNA for efficient gene transfer and restoration of p53 levels in cancer cells.
Area of Science:
- Molecular Biology
- Biotechnology
- Cancer Research
Background:
- The p53 tumor suppressor is crucial for preventing malignant cell transformation.
- Effective gene therapy relies on efficient gene transfer, which is influenced by vector manufacturing.
- High-purity supercoiled plasmid DNA is essential for pharmaceutical applications.
Purpose of the Study:
- To describe a method for preparing and purifying supercoiled plasmid DNA to meet regulatory standards.
- To evaluate the efficacy of arginine affinity chromatography for plasmid DNA purification.
- To demonstrate the biological activity of purified plasmid DNA in cancer cell lines.
Main Methods:
- Utilized arginine affinity chromatography for plasmid DNA purification.
- Developed detailed procedures for downstream processing of plasmid DNA.
- Assessed the purity and quality of the supercoiled plasmid DNA.
Main Results:
- Achieved pharmaceutical-grade purity of supercoiled plasmid DNA.
- The purified plasmid DNA demonstrated efficient transfection in eukaryotic cells.
- Restoration of p53 protein levels was observed in treated cancer cell lines.
Conclusions:
- Arginine affinity chromatography is a viable method for producing high-purity plasmid DNA.
- The purified plasmid DNA is biologically active and suitable for gene therapy applications.
- This approach offers a promising strategy for effective anticancer treatment through p53 reestablishment.
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