Selective purification of supercoiled plasmid DNA from clarified cell lysates with a single histidine-agarose chromatography step.
The ability to isolate sc (supercoiled) pDNA (plasmid DNA) isoform should be one of the features of a pDNA purification process, eliminating sample contaminants such as RNA, gDNA (genomic DNA), proteins and endotoxins. A process is described that uses a single histidine-agarose chromatography step to purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. The histidine-agarose support combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. The 6 kb DNA vaccine backbone pVAX1-LacZ was used as a model target. Following loading at high salt [2.3 M (NH4)2SO4], the different species were eluted by a series of reverse salt step gradients (2.0, 1.5 and 0 M (NH4)2SO4). Open circular pDNA and gDNA was eluted at 2.3 M, sc pDNA was isolated as a single peak at 2.0 M and RNA was eluted at 1.5 M (NH4)2SO4 and lower. The underlying mechanism is thought to involve not only hydrophobic interactions between the support and pDNA molecules, but also non-specific biorecognition of nucleic acid bases by the histidine ligand. Control analysis showed that the isolated sc pDNA conforms to specifications in terms of gDNA (3.4 ng/microg of pDNA), endotoxins (0.02 endotoxin unit/microg of pDNA), RNA and proteins (undetectable) and pDNA homogeneity (approximately 100% sc). Furthermore, the transfection efficiency of Chinese-hamster ovary cells (50%) was significantly higher when compared with the efficiency (25%) of a pDNA control. The present study confirms the possibility of using a single histidine-agarose chromatography step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.
Sousa
Fani
F
Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
Freitas
Sindélia
S
Azzoni
Adriano R
AR
Prazeres
Duarte Miguel F
DM
Queiroz
João
J
D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification*
D004278:DNA, Superhelical; D006639:Histidine