Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions.
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.
O'Mahony
Kevin
K
Laboratory of Chemical Biotechnology, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Freitag
Ruth
R
Hilbrig
Frank
F
Schumacher
Ivo
I
Müller
Patrick
P
D001419:Bacteria / Q000378:metabolism*; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004269:DNA, Bacterial; D005374:Filtration; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007221:Industry; D009113:Muramidase / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism*; D013696:Temperature; D014325:Tromethamine / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D004269:DNA, Bacterial; D014325:Tromethamine; D014508:Urea; D004247:DNA; D009113:Muramidase; D019791:Guanidine