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Plasmid purification by using a new naphthalene tripodal support

2017-06-30Journal article Open access
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dc.contributor.authorSantos, Tiago
dc.contributor.authorProença, Z.
dc.contributor.authorQueiroz, João
dc.contributor.authorTomaz, C. T.
dc.contributor.authorCruz, Carla
dc.date.accessioned2020-02-07T16:41:58Z
dc.date.available2022-06-30T00:30:17Z
dc.date.issued2017-06-30
dc.description.abstractThe aim of this work was to employ a new naphthalene tripodal support for the isolation of supercoiled (sc) isoform of plasmid (pDNA) from a native sample. This support is for the first time synthesized and used in pDNA purification. The naphthalene tripodal ligand was synthesized and characterized to assess its purity and subsequently immobilized onto an epoxy-activated Sepharose CL-6B, using mild conditions and resulting in a ligand density of 0.32 mmol naphthalene tripodal/g derivatized Sepharose CL-6B. The complete characterization of naphthalene tripodal Sepharose CL-6B support was performed by High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. The affinity was measured by SPR biosensor between naphthalene tripodal ligand immobilized on the surface and sc pVAX1-LacZ and the KD was 8.65 10 8 ± 1.0 10 8 M in 10 mM Tris-HCl pH 8.0, at T = 25 C, indicating a high affinity. For comparison reasons, the affinity ligand 3,8-diamino-6-phenylphe nanthridine (DAPP) was also immobilized on the chip surface and the KD for sc pVAX1-LacZ is lower than with naphthalene tripodal. Saturation transfer difference-nuclear magnetic resonance spectroscopy (STD-NMR) experiments showed that the interactions between the naphthalene tripodal–Sepharose CL-6B and DAPP-Sepharose supports and the 50-mononucleotides are mainly hydrophobic and p-p stacking. The isolation of sc pDNA isoform was achieved with low salt concentrations, using 95 mM NaCl in binding step and 550 mM NaCl in elution step at T = 4 C and pH 8, thus reducing the economic and environmental impact.pt_PT
dc.description.sponsorshipProject POCI-01-0145-FEDER-007491; fellowship from Santander/Totta-UBI (Ref BID/ICI-FCS/CICS/Santander Universidades-UBI/2016).pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1016/j.seppur.2017.06.072pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/9144
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.subjectNaphthalene tripodal supportpt_PT
dc.subjectAffinity chromatographypt_PT
dc.subjectHR-MAS NMR spectroscopypt_PT
dc.subjectSupercoiled plasmid purificationpt_PT
dc.titlePlasmid purification by using a new naphthalene tripodal supportpt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/COMPETE/EXPL%2FQEQ-MED%2F1068%2F2013/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/5876/UID%2FMulti%2F00709%2F2013/PT
oaire.citation.endPage89pt_PT
oaire.citation.startPage81pt_PT
oaire.citation.titleSeparation and Purification Technologypt_PT
oaire.citation.volume188pt_PT
oaire.fundingStreamCOMPETE
oaire.fundingStream5876
person.familyNameAfonso dos Santos
person.familyNameQueiroz
person.familyNameTomaz
person.familyNameCruz
person.givenNameTiago André
person.givenNameJoão
person.givenNameCândida
person.givenNameCarla
person.identifier2198714
person.identifier.ciencia-id6517-B7D0-2B07
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-idFE13-0C44-13D1
person.identifier.ciencia-id6816-7888-F8B1
person.identifier.orcid0000-0003-4620-0174
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0002-0927-2331
person.identifier.orcid0000-0001-6630-1242
person.identifier.ridL-3104-2014
person.identifier.ridF-9367-2014
person.identifier.ridI-7806-2013
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id7004546303
person.identifier.scopus-author-id7202701394
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.rightsopenAccesspt_PT
rcaap.typearticlept_PT
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