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

2019Journal article Restricted access
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dc.contributor.authorValente, Joana
dc.contributor.authorSousa, A.
dc.contributor.authorQueiroz, João
dc.contributor.authorSousa, Fani
dc.date.accessioned2020-01-10T10:04:03Z
dc.date.available2020-01-10T10:04:03Z
dc.date.issued2019
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 chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. 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 endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1016/j.jchromb.2018.12.002pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/8189
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S1570023218307153pt_PT
dc.subjectChromatography - Affinitypt_PT
dc.subjectDNA - Recombinantpt_PT
dc.subjectDNA - Superhelicalpt_PT
dc.subjectEscherichia colipt_PT
dc.subjectPlasmidspt_PT
dc.subjectReproducibility of Resultspt_PT
dc.subjectResearch Designpt_PT
dc.subjectTumor Suppressor Protein p53pt_PT
dc.subjectTyrosinept_PT
dc.titleDoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatographypt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F96809%2F2013/PT
oaire.citation.endPage192pt_PT
oaire.citation.startPage184pt_PT
oaire.citation.titleJournal of Chromatography Bpt_PT
oaire.citation.volume1105pt_PT
oaire.fundingStreamSFRH
person.familyNameValente
person.familyNameQueiroz
person.familyNameSousa
person.givenNameJoana
person.givenNameJoão
person.givenNameFani
person.identifierVABrK9AAAAAJ
person.identifier.ciencia-id7515-7468-212D
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-id991D-2E13-A840
person.identifier.orcid0000-0002-2408-9762
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0001-9996-2194
person.identifier.ridL-3104-2014
person.identifier.ridA-2014-2017
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id7005110268
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.embargofctCopyright cedido à editora no momento da publicaçãopt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublicationd18cb0c2-0e40-40fe-a2dc-429c8a3ccb0b
relation.isAuthorOfPublicationc798c033-c160-4f99-a8ad-04416923943f
relation.isAuthorOfPublication2935de97-fc90-4d38-9505-4caa364f8a10
relation.isAuthorOfPublication.latestForDiscoveryd18cb0c2-0e40-40fe-a2dc-429c8a3ccb0b
relation.isProjectOfPublication87b297e6-680f-4f91-8816-b81db0a917bd
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