Entwicklung und Untersuchung eines Verfahrens zur integrierten Aufreinigung von Plasmid DNA mittels wässriger Zweiphasenextraktion

Plasmids are double stranded circular DNA molecules, mainly known as vectors for transferring genes. Therefore, plasmids have a high importance for molecular biology and gene technology respectively. Presently, there is a high interest to utilize plasmids in gene therapy and DNA vaccination due to their ability to replace or supplement genes. The manufacturing of plasmid DNA is performed using biotechnological processes by cultivation of optimized E. coli strains. The developed three-step purification process begins with an alkaline lysis of bacterial cells and separation of crude impurities and cell debris using two phase extraction. In this study, a systematic screening led to an optimal system comprising 15% PEG 800 and 20% potassium phosphate. This system resulted in a plasmid DNA recovery of about 90 % plasmid in the bottom phase, while most of contaminants together with solids (cell precipitate) are separated into the top phase. Partitioning experiments of plasmid DNA and RNA, performed as a function of temperature, pH and PEG molecular weight, showed a close relation between system conditions and partitioning of plasmid DNA. In the investigated two-phase system it was found that changing the temperature from 15 to 20°C leads to a partitioning of plasmid DNA from top to bottom phase. This could be attributed to the plasmid solubility in the top phase, which is drastically affected by variation of PEG molecular weight and temperature respectively. The assumption is, that differences of solubility are associated with a structural change of plasmid DNA, known as coil-globule transition. After separation of the main contaminants (capturing) and extraction of plasmid DNA into the bottom phase, the plasmid DNA is isolated from the bottom phase using an adsorptive membrane step. The high potassium phosphate concentrations in the bottom phase were exploited in order to facilitate hydrophobic interaction for the adsorption of plasmid DNA on a nylon membrane followed by a subsequent elution using TE-buffer. During this desalting and concentration process, a step recovery of 95% plasmid DNA and an 85% reduction of the RNA content could be determined. For a specific depletion of chromosomal DNA and open circular plasmid DNA a third process step was developed, which is based on a sequence comprising denaturation of the nucleic acids present and selective renaturation of supercoiled plasmid DNA followed by selective partitioning in an ATPS. During this procedure, chromosomal and open circular DNA are irreversibly denatured. The drastic change of hydrophobicity enabled the separation from native supercoiled plasmid DNA using the second aqueous two phase system. With this procedure, the fraction of open circular DNA is reduced to 4-6% and of chromosomal DNA to <1% together with a supercoiled DNA yield of 90-95%. The complete process for purification of plasmid DNA is thus a combination of a first extraction for the separation of main impurities (capture), followed by desalting step using reversible membrane adsorption and polish step for the removal of chromosomal DNA and open circular plasmids. Consequently, this procedure was applied for the processing of 100 g biomass and compared to the chromatography based purification method established and commercialized by QIAGEN. As a result, comparable recoveries were achieved, whereas the fraction of supercoiled plasmids was slightly higher for the extraction procedure. Regarding the fraction of chromosomal DNA, open circular plasmids and RNA, the targeted specifications were met. A further scale up of the process seems feasible after solving limitations connected with the mechanical process of phase separation. In addition, extraction in small scale by combining extraction with membrane adsorption shows an interesting alternative for mini-preparation of plasmid DNA.

Grantor dc:publisher

Forschungszentrum, Zentralbibliothek

Year dc:date

2006

Author dc:creator

• Frerix, Andreas

Contributors dc:contributor

• Wandrey, Christian

• info:eu-repo/classification/ddc/620
• Ingenieurwissenschaften
• Plasmid
• DNS
• Extraktion
• Aufreinigung
• Produktaufarbeitung
• wässriges Zweiphasensystem
• supercoiled
• ATPS
• downstream processing
• DNA

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123471%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61851