A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells.
Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s(-1), with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed.
Ciccolini
Laura A S
LA
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE UK.
Shamlou
Parviz Ayazi
PA
Titchener-Hooker
Nigel
N
D001433:Bacteriolysis / Q000235:genetics / Q000502:physiology; D019149:Bioreactors; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004278:DNA, Superhelical; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000096:biosynthesis* / Q000302:isolation & purification*; D012972:Sodium Hydroxide / Q000032:analysis; D013053:Spectrophotometry; D013314:Stress, Mechanical; D013997:Time Factors; D014783:Viscosity
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012972:Sodium Hydroxide