Plasmid purification by using a new naphthalene tripodal support

Detalhes bibliográficos
Autor(a) principal: Santos, Tiago
Data de Publicação: 2017
Outros Autores: Proença, Z., Queiroz, João, Tomaz, C. T., Cruz, Carla
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10400.6/9144
Resumo: The 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.
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spelling Plasmid purification by using a new naphthalene tripodal supportNaphthalene tripodal supportAffinity chromatographyHR-MAS NMR spectroscopySupercoiled plasmid purificationThe 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.Project POCI-01-0145-FEDER-007491; fellowship from Santander/Totta-UBI (Ref BID/ICI-FCS/CICS/Santander Universidades-UBI/2016).ElsevieruBibliorumSantos, TiagoProença, Z.Queiroz, JoãoTomaz, C. T.Cruz, Carla2022-06-30T00:30:17Z2017-06-302017-06-30T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.6/9144eng10.1016/j.seppur.2017.06.072info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-12-15T09:49:54Zoai:ubibliorum.ubi.pt:10400.6/9144Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:49:21.911639Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Plasmid purification by using a new naphthalene tripodal support
title Plasmid purification by using a new naphthalene tripodal support
spellingShingle Plasmid purification by using a new naphthalene tripodal support
Santos, Tiago
Naphthalene tripodal support
Affinity chromatography
HR-MAS NMR spectroscopy
Supercoiled plasmid purification
title_short Plasmid purification by using a new naphthalene tripodal support
title_full Plasmid purification by using a new naphthalene tripodal support
title_fullStr Plasmid purification by using a new naphthalene tripodal support
title_full_unstemmed Plasmid purification by using a new naphthalene tripodal support
title_sort Plasmid purification by using a new naphthalene tripodal support
author Santos, Tiago
author_facet Santos, Tiago
Proença, Z.
Queiroz, João
Tomaz, C. T.
Cruz, Carla
author_role author
author2 Proença, Z.
Queiroz, João
Tomaz, C. T.
Cruz, Carla
author2_role author
author
author
author
dc.contributor.none.fl_str_mv uBibliorum
dc.contributor.author.fl_str_mv Santos, Tiago
Proença, Z.
Queiroz, João
Tomaz, C. T.
Cruz, Carla
dc.subject.por.fl_str_mv Naphthalene tripodal support
Affinity chromatography
HR-MAS NMR spectroscopy
Supercoiled plasmid purification
topic Naphthalene tripodal support
Affinity chromatography
HR-MAS NMR spectroscopy
Supercoiled plasmid purification
description The 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.
publishDate 2017
dc.date.none.fl_str_mv 2017-06-30
2017-06-30T00:00:00Z
2022-06-30T00:30:17Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
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status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10400.6/9144
url http://hdl.handle.net/10400.6/9144
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1016/j.seppur.2017.06.072
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
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dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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