Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support

Abstract

This work was supported by FEDER funds through the POCI - COMPETE 2020 - Operational Programme Competitive- ness and Internationalization in Axis I - Strengthening research, technological development and innovation (Project POCI-01-0145- FEDER-007491) and National Funds by FCT - Foundation for Sci- ence and Technology (Project UID/Multi/00709/2019 ). J.F.A. Valente and A. Sousa also acknowledge Ph.D. and Postdoctoral fellowships (Ref SFRH/BD/96809/2013 and Ref SFRH/BPD/102716/2014, respec- tively).p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alter- native to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA pu- rification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macrop- orous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic fea- sibility in case of scale-up.info:eu-repo/semantics/publishedVersio