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Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands

2017Journal article Restricted access
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dc.contributor.authorAmorim, Lúcia
dc.contributor.authorGaspar, Rita
dc.contributor.authorPereira, Patrícia
dc.contributor.authorČernigoj, Urh
dc.contributor.authorSousa, Fani
dc.contributor.authorQueiroz, João
dc.contributor.authorSousa, Ângela
dc.date.accessioned2020-01-09T15:41:47Z
dc.date.available2020-01-09T15:41:47Z
dc.date.issued2017
dc.description.abstractAffinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1002/elps.201700147pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/8172
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherWileypt_PT
dc.relationPlatform for the biosynthesis and purification of HPV-16 E6/E7mutant DNA vaccine for cervical cancer treatment or prevention
dc.relation.publisherversionhttps://onlinelibrary.wiley.com/doi/full/10.1002/elps.201700147pt_PT
dc.subjectHistidine-based ligandspt_PT
dc.subjectMonolithspt_PT
dc.subjectPlasmid DNA purificationpt_PT
dc.subjectRNA removalpt_PT
dc.subjectSample displacement chromatographypt_PT
dc.titleChromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligandspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardTitlePlatform for the biosynthesis and purification of HPV-16 E6/E7mutant DNA vaccine for cervical cancer treatment or prevention
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F81914%2F2011/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT//SFRH%2FBPD%2F102716%2F2014/PT
oaire.citation.endPage2980pt_PT
oaire.citation.issue22-23pt_PT
oaire.citation.startPage2975pt_PT
oaire.citation.titleELECTROPHORESISpt_PT
oaire.citation.volume38pt_PT
oaire.fundingStreamSFRH
person.familyNameAlves Amorim
person.familyNamePereira
person.familyNameSousa
person.familyNameQueiroz
person.familyNameSousa
person.givenNameLúcia Filipa
person.givenNamePatrícia
person.givenNameFani
person.givenNameJoão
person.givenNameÂngela
person.identifier817754
person.identifier.ciencia-id7517-14AE-F5A3
person.identifier.ciencia-id4715-067B-4FD0
person.identifier.ciencia-id991D-2E13-A840
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-idFB1F-08DE-23FE
person.identifier.orcid0000-0002-6078-4922
person.identifier.orcid0000-0002-5665-5271
person.identifier.orcid0000-0001-9996-2194
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0001-9155-7581
person.identifier.ridA-2014-2017
person.identifier.ridL-3104-2014
person.identifier.scopus-author-id56111844900
person.identifier.scopus-author-id7005110268
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id24330268800
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
rcaap.embargofctCopyright cedido à editora no momento da publicaçãopt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
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