Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography
Autor(a) principal: | |
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Data de Publicação: | 2018 |
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/10400.6/8184 |
Resumo: | The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages. |
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Arginine and di-arginine ligands for plasmid DNA purification using negative chromatographyPlasmid DNA purificationPlasmid DNA purificationArginineArginineAgarose resinThe increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages.This work was supported by CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico (grants 444412/2014-0, 304906/2014-0 and 307739/2015-5), and FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo (grant 2013/23780-1). The authors acknowledges to Professor Miguel Prazeres, from Instituto Superior Técnico, Lisboa, Portugal, for the kindly donation of the E. coli DH10b harboring the pVAX1-GFP plasmid.ElsevieruBibliorumCardoso, SaraFilho, Pedro De Alcântara PessôaSousa, FaniAzzoni, Adriano2020-01-09T17:04:23Z20182018-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.6/8184eng10.1016/j.seppur.2018.03.066metadata only accessinfo: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:RCAAP2023-03-29T02:31:14ZPortal AgregadorONG |
dc.title.none.fl_str_mv |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
title |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
spellingShingle |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography Cardoso, Sara Plasmid DNA purification Plasmid DNA purification Arginine Arginine Agarose resin |
title_short |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
title_full |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
title_fullStr |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
title_full_unstemmed |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
title_sort |
Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography |
author |
Cardoso, Sara |
author_facet |
Cardoso, Sara Filho, Pedro De Alcântara Pessôa Sousa, Fani Azzoni, Adriano |
author_role |
author |
author2 |
Filho, Pedro De Alcântara Pessôa Sousa, Fani Azzoni, Adriano |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
uBibliorum |
dc.contributor.author.fl_str_mv |
Cardoso, Sara Filho, Pedro De Alcântara Pessôa Sousa, Fani Azzoni, Adriano |
dc.subject.por.fl_str_mv |
Plasmid DNA purification Plasmid DNA purification Arginine Arginine Agarose resin |
topic |
Plasmid DNA purification Plasmid DNA purification Arginine Arginine Agarose resin |
description |
The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018 2018-01-01T00:00:00Z 2020-01-09T17:04:23Z |
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/10400.6/8184 |
url |
http://hdl.handle.net/10400.6/8184 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1016/j.seppur.2018.03.066 |
dc.rights.driver.fl_str_mv |
metadata only access info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
metadata only access |
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 |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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1777301827785588736 |