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Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Title data

O'Mahony, Kevin ; Freitag, Ruth ; Hilbrig, Frank ; Schumacher, Ingo ; Müller, Patrick:
Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions.
In: Biotechnology Progress. Vol. 23 (2007) Issue 4 . - pp. 895-903.
ISSN 1520-6033
DOI: https://doi.org/10.1021/bp0701113

Abstract in another language

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.

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: 600 Technology, medicine, applied sciences
600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited: 24 Feb 2016 15:19
Last Modified: 24 Feb 2016 15:19
URI: https://eref.uni-bayreuth.de/id/eprint/31043