Please use this identifier to cite or link to this item: http://hdl.handle.net/1822/3424

Full metadata record
DC FieldValueLanguage
dc.contributor.authorTrindade, Inês P.-
dc.contributor.authorDiogo, Maria M.-
dc.contributor.authorPrazeres, Duarte M. F.-
dc.contributor.authorMarcos, João Carlos-
dc.date.accessioned2005-11-07T10:43:22Z-
dc.date.available2005-11-07T10:43:22Z-
dc.date.issued2005-
dc.identifier.citation"Journal of Chromatography A". ISSN 0021-9673. 1082 (2005) 176-184.eng
dc.identifier.issn0021-9673eng
dc.identifier.urihttp://hdl.handle.net/1822/3424-
dc.description.abstractThe current study explores the possibility of using a polyethyleneglycol(PEG)/ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400, 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20 % (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16 % w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35 % w/w PEG 600, 6% w/w NH4)2SO4), it was possible to recover 100 % of pDNA in the bottom phase with a 3-fold increase in concentration. Further increase in the lysate load up to 40 %(w/w) with this system resulted in a 8-fold increase in pDNA concentration, but with a yield loss of 15 %. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with isopropanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.eng
dc.description.sponsorshipPortuguese Ministry of Science and Technology - POCTI/BIO/47245/2002.por
dc.language.isoengeng
dc.publisherElseviereng
dc.rightsopenAccesseng
dc.subjectAqueous two-phase systemseng
dc.subjectLiquid-liquid extractioneng
dc.subjectPlasmid DNA purificationeng
dc.subjectPolyethylene glycoleng
dc.subjectGene therapyeng
dc.subjectAmmonium sulphateeng
dc.subjectpurificationpor
dc.subjectplasmidpor
dc.subjectDNApor
dc.titlePurification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatographyeng
dc.typearticlepor
dc.peerreviewedyeseng
dc.relation.publisherversionhttp://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TG8-4GDSF0P-1-D&_cdi=5248&_user=2459786&_orig=browse&_coverDate=08%2F05%2F2005&_sk=989179997&view=c&wchp=dGLzVzz-zSkWA&md5=d515cd055cd9a2ad6f9f005d425781fd&ie=/sdarticle.pdfeng
sdum.pagination176-184eng
sdum.publicationstatuspublishedeng
sdum.volume1082eng
oaire.citationStartPage176por
oaire.citationEndPage184por
oaire.citationIssue2por
oaire.citationVolume1082por
dc.identifier.doi10.1016/j.chroma.2005.05.079por
dc.identifier.pmid16035359por
dc.subject.wosScience & Technologypor
sdum.journalJournal of Chromatography Apor
Appears in Collections:CDQuim - Artigos (Papers)

Files in This Item:
File Description SizeFormat 
ArtPlasmPEGNH4SO4_RepUM.pdfDocumento principal161,61 kBAdobe PDFView/Open
ArtPlasmPEGNH4SO4_Figures_RepUM.pdfFiguras618,39 kBAdobe PDFView/Open
ArtPlasmPEGNH4SO4_Tables_RepUM.pdfTabelas56,36 kBAdobe PDFView/Open

Partilhe no FacebookPartilhe no TwitterPartilhe no DeliciousPartilhe no LinkedInPartilhe no DiggAdicionar ao Google BookmarksPartilhe no MySpacePartilhe no Orkut
Exporte no formato BibTex mendeley Exporte no formato Endnote Adicione ao seu ORCID