Plasmid purification by using a new naphthalene tripodal support
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , |
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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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 |
format |
article |
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 |
dc.format.none.fl_str_mv |
application/pdf |
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) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799136385899167744 |