Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate.
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production.
Wahlund
P-O
PO
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Lund, Sweden.
Gustavsson
P-E
PE
Izumrudov
V A
VA
Larsson
P-O
PO
Galaev
I Yu
IY
D000042:Absorption; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004270:DNA, Circular / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D009696:Nucleic Acids / Q000032:analysis / Q000737:chemistry; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011095:Polyethylenes / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry
D004270:DNA, Circular; D009696:Nucleic Acids; D011073:Polyamines; D000071228:Polyelectrolytes; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride