Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations.
Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.
Merck & Co., Inc., P.O. Box 2000, R8OY-3A42, Rahway, New Jersey 07065-0900, USA. francis_meacle@merck.com
F J
FJ
Meacle
R
R
Lander
P
P
Ayazi Shamlou
N J
NJ
Titchener-Hooker
D001698:Biomedical Engineering / Q000379:methods; D002457:Cell Extracts / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D033081:Shear Strength; D012972:Sodium Hydroxide / Q000737:chemistry
D002457:Cell Extracts; D004269:DNA, Bacterial; D012972:Sodium Hydroxide