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Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale.

Title data

O'Mahony, Kevin ; Freitag, Ruth ; Hilbrig, Frank ; Müller, Patrick ; Schumacher, Ivo:
Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale.
In: Journal of Biotechnology. Vol. 119 (September 2005) Issue 2 . - pp. 118-132.
ISSN 1873-4863
DOI: https://doi.org/10.1016/j.jbiotec.2005.03.020

Official URL: Volltext

Abstract in another language

The paper addresses the question of how to achieve bacterial lysis in large-scale plasmid DNA production processes, where conventional alkaline lysis may become awkward to handle. Bacteria were grown in shaker flasks and a bioreactor. Suboptimal growth conditions were found advantageous for stable plasmid production at high copy numbers (up to 25mg/L could be achieved). Cells were harvested by filtration in the presence of a filter aid. A linear relationship between the biomass and the optimal filter aid concentration in terms of back pressure could be established. Bacteria-containing filter cakes were washed with isotonic buffer and lysis was achieved in situ by a two-step protocol calling for fragilisation of the cells followed by heat lysis in a suitable buffer. RNA and other soluble cell components where washed out of the cake during this step, while the plasmid DNA was retained. Afterwards a clear lysate containing relatively pure plasmid DNA could be eluted from the cake mostly as the desired supercoiled topoisomer, while cell debris and genomic DNA were retained. Lysis is, thus, integrated not only with cell capture but also with a significant degree of isolation/purification, as most impurities were considerably reduced during the procedure.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag
Faculties
Faculties > Faculty of Engineering Science
Faculties > Faculty of Engineering Science > Chair Process Biotechnology
Result of work at the UBT: No
DDC Subjects: 500 Science
600 Technology, medicine, applied sciences
600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited: 25 Feb 2016 14:23
Last Modified: 25 Feb 2016 14:23
URI: https://eref.uni-bayreuth.de/id/eprint/31055