Circular dichroism investigation of the effect of plasmid DNA structure on retention in histidine chromatography |
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Authors: | Sousa F Prazeres D M F Queiroz J A |
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Institution: | a CICS-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal b IBB, Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, 1049-001 Lisboa, Portugal |
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Abstract: | The effect of temperature-induced changes in secondary and tertiary structures of plasmid DNA (pDNA) and on the retention behaviour of open circular (oc) and supercoiled (sc) isoforms in histidine-agarose chromatography was investigated by Circular dichroism (CD) spectroscopy. Chromatographic experiments performed with three plasmids (2.7, 6.1 and 7.4 kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0 M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 °C. Such behaviour was attributed to the temperature-induced removal of negative superhelical turns in sc pDNA which is accompanied by a decrease in the number of dissociated base pairs responsible for interaction with the histidine ligands. CD spectroscopy showed that temperature has an important effect on plasmid secondary structure if adenine-rich inserts are present in the plasmid structure. Chromatographic experiments also suggested that base composition could also be responsible for the induction of specific interactions with histidine ligands. |
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Keywords: | Circular dichroism Histidine-chromatography Plasmid DNA structure Supercoiling |
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