Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale.
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.
Laboratory of Chemical Biotechnology, Faculty of Basic Science, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Kevin
K
O'Mahony
Ruth
R
Freitag
Frank
F
Hilbrig
Patrick
P
Müller
Ivo
I
Schumacher
D001433:Bacteriolysis; D018533:Biomass; D002021:Buffers; D002469:Cell Separation; D005591:Chemical Fractionation; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004270:DNA, Circular / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005374:Filtration; D006358:Hot Temperature; D009113:Muramidase; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D013347:Subcellular Fractions; D013696:Temperature
D002021:Buffers; D004269:DNA, Bacterial; D004270:DNA, Circular; D004278:DNA, Superhelical; D009113:Muramidase