Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification.
New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.
Sousa
F
F
CICS, Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
Prazeres
D M F
DM
Queiroz
J A
JA
D001120:Arginine / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D012685:Sepharose / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry
D004278:DNA, Superhelical; D012965:Sodium Chloride; D012685:Sepharose; D001120:Arginine