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DoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatography

2018-12-15Journal article Open access
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dc.contributor.authorValente, J.F.A.
dc.contributor.authorSousa, A.
dc.contributor.authorQueiroz, J.A.
dc.contributor.authorSousa, F.
dc.date.accessioned2023-05-31T13:27:33Z
dc.date.available2023-05-31T13:27:33Z
dc.date.issued2018-12-15
dc.description.abstractP53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O phospho-L-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chro matographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-en coding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was ap plied. The dynamic binding capacity of the O-phospho-L-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/ mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of en dotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agenciespt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationJ.F.A. Valente, A. Sousa, J.A. Queiroz, F. Sousa, DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography, Journal of Chromatography B, Volume 1105, 2019, Pages 184-192, ISSN 1570-0232, https://doi.org/10.1016/j.jchromb.2018.12.002pt_PT
dc.identifier.doi10.1016/j.jchromb.2018.12.002pt_PT
dc.identifier.issn1570-0232
dc.identifier.urihttp://hdl.handle.net/10400.8/8546
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S1570023218307153pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/pt_PT
dc.subjectSupercoiled p53-encoding plasmidpt_PT
dc.subjectComposite Central Face designpt_PT
dc.subjectO-Phospho-L-tyrosine chromatographypt_PT
dc.subjectDesign of Experimentspt_PT
dc.titleDoE to improve supercoiled p53-pDNA purification by O-phospho-L-tyrosine chromatographypt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage192pt_PT
oaire.citation.startPage184pt_PT
oaire.citation.titleJournal of Chromatography Bpt_PT
oaire.citation.volume1105pt_PT
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
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublication7a9941e0-69b5-48f5-9068-c950fb064a4f
relation.isAuthorOfPublication.latestForDiscovery7a9941e0-69b5-48f5-9068-c950fb064a4f

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