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Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

2019-11-28Journal article Open access
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dc.contributor.authorAzevedo, G.M.
dc.contributor.authorValente, J.F.A.
dc.contributor.authorSousa, A.
dc.contributor.authorPedro, A.Q.
dc.contributor.authorPereira, P.
dc.contributor.authorSousa, F.
dc.contributor.authorQueiroz, J.A.
dc.date.accessioned2023-04-05T15:32:19Z
dc.date.available2023-04-05T15:32:19Z
dc.date.issued2019-11-28
dc.descriptionFunding: This work was supported by FEDER funds through the POCI—COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi /00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.pt_PT
dc.descriptionAcknowledgments: The authors would like to thank Thomas Roberts for providing the pcDNA3–FLAG–p53 construct through Addgene, ref: 10838.pt_PT
dc.description.abstractThe dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationAzevedo GM, Valente JFA, Sousa A, Pedro AQ, Pereira P, Sousa F, Queiroz JA. Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity. Applied Sciences. 2019; 9(23):5170. https://doi.org/10.3390/app9235170pt_PT
dc.identifier.doihttps://doi.org/10.3390/app9235170pt_PT
dc.identifier.eissn2076-3417
dc.identifier.other5170
dc.identifier.urihttp://hdl.handle.net/10400.8/8337
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherMDPIpt_PT
dc.relationHealth Sciences Research Centre
dc.relationCICECO-Aveiro Institute of Materials
dc.relationAffinity purification and cell-specific delivery of a p53-encoding plasmid DNA for gene mediated cancer therapy
dc.relation.publisherversionhttps://www.mdpi.com/2076-3417/9/23/5170pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectAffnity chromatographypt_PT
dc.subjectChromatographic conditionspt_PT
dc.subjectp53 encoding plasmidpt_PT
dc.subjectSupercoiled pDNA bioactivitypt_PT
dc.subjectGene-based cancer therapypt_PT
dc.titleEffect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivitypt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitleHealth Sciences Research Centre
oaire.awardTitleCICECO-Aveiro Institute of Materials
oaire.awardTitleAffinity purification and cell-specific delivery of a p53-encoding plasmid DNA for gene mediated cancer therapy
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID%2FMulti%2F00709%2F2013/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID%2FCTM%2F50011%2F2019/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/FARH/SFRH%2FBD%2F96809%2F2013/PT
oaire.citation.endPage20pt_PT
oaire.citation.issue23pt_PT
oaire.citation.startPage1pt_PT
oaire.citation.titleApplied Sciencespt_PT
oaire.citation.volume9pt_PT
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStreamFARH
person.familyNameValente
person.givenNameJoana
person.identifier826126
person.identifier.ciencia-id7515-7468-212D
person.identifier.orcid0000-0002-2408-9762
person.identifier.ridM-1930-2019
person.identifier.scopus-author-id56273710100
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
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relation.isAuthorOfPublication.latestForDiscovery7a9941e0-69b5-48f5-9068-c950fb064a4f
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