Dynamic binding capacity of plasmid DNA in histidine-agarose chromatography.
The use of histidine-agarose chromatography in the purification of supercoiled (sc) plasmid DNA (pDNA) from Escherichia coli lysates has been reported recently. In the current work we describe a set of breakthrough experiments which were designed to study the effect of parameters such as flow-rate, temperature, concentration and conformation on the dynamic binding capacity of pDNA to the histidine support. One of the most striking results shows that the dynamic binding capacity for sc pDNA decreases linearly from 250.8 to 192.0 microg sc pDNA/mL when the temperature is varied from 5 to 24 degrees C. This behaviour was attributed to temperature-induced, pre-denaturation conformational changes which promote the removal of negative superhelical turns in sc pDNA molecules and decrease the interaction of DNA bases with the histidine ligands. The capacity for sc pDNA was highly improved when using feeds with higher pDNA concentrations, a phenomenon which was attributed to the fact that pDNA molecules in more concentrated solutions are significantly compressed. A maximum capacity of 530.0 microg pDNA/mL gel was obtained when using a 125 microg/mL pDNA feed at 1 mL/min and 5 degrees C, a figure which is comparable to the plasmid capacity values published for other chromatographic supports. Finally, a more than 2-fold increase in capacity was obtained when changing from open circular to sc pDNA solutions. Overall, the results obtained provide valuable information for the future development and implementation of histidine chromatography in the process scale purification of pDNA.
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
D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D010957:Plasmids; D012685:Sepharose / Q000737:chemistry*; D013696:Temperature
D006639:Histidine; D004247:DNA; D012685:Sepharose