Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction.
The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery.
Tschapalda
Kirsten
K
Chair of Fermentation Engineering, Faculty of Technology, Bielefeld University, 33501 Bielefeld, Germany.
Streitner
Nadine
N
Voss
Carsten
C
Flaschel
Erwin
E
D000468:Alkalies / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D008823:Micelles; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification
D000468:Alkalies; D004269:DNA, Bacterial; D008823:Micelles