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Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification
Author(s) -
Sousa F.,
Prazeres D. M. F.,
Queiroz J. A.
Publication year - 2009
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1097
Subject(s) - chemistry , elution , affinity chromatography , arginine , chromatography , nucleic acid , dna , gene isoform , amino acid , plasmid , biochemistry , gene , enzyme
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 m m ) or arginine (20–70 m m ) 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. Copyright © 2008 John Wiley & Sons, Ltd.