Please use this identifier to cite or link to this item: http://hdl.handle.net/1822/23076

TitlePurification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography
Author(s)Nunes, C. Caramelo
Gabriel, M. F.
Almeida, Paulo
Marcos, João Carlos
Tomaz, C. T.
KeywordsPlasmid purification
Small DNA ligands
Berenil
Pseudo-affinity chromatography
Issue date1-Sep-2012
PublisherElsevier
JournalJournal of Chromatography B
Abstract(s)In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3 M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0 M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.
TypeArticle
URIhttp://hdl.handle.net/1822/23076
DOI10.1016/j.jchromb.2012.07.023
ISSN1570-0232
Publisher versionhttp://www.sciencedirect.com/
Peer-Reviewedyes
AccessOpen access
Appears in Collections:CDQuim - Artigos (Papers)

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