Dynamic binding capacity of plasmid DNA in histidine–agarose chromatography

Abstract

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.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

“…For adsorption of scpDNA to particle-based chromatographic supports (e.g. histidine-agarose chromatography [25]), the dynamic binding capacity of pDNA molecules was found to decrease at higher feed flowrates. This is presumably due to a combination of the extension of pDNA molecules (by high flowrate driven external hydrodynamic forces) [21] and shorter time allowed for pDNA to ligand interaction at high flowrate which enabled fewer molecules to access the ligands on the resin (especially for the ligands in the intraparticle pores) [25].…”

“…histidine-agarose chromatography [25]), the dynamic binding capacity of pDNA molecules was found to decrease at higher feed flowrates. This is presumably due to a combination of the extension of pDNA molecules (by high flowrate driven external hydrodynamic forces) [21] and shorter time allowed for pDNA to ligand interaction at high flowrate which enabled fewer molecules to access the ligands on the resin (especially for the ligands in the intraparticle pores) [25]. On the other hand, adsorption of pDNA to anion-exchange DEAE (diethylaminoethyl) monolith column was reported to be flowrateindependent from 120 to 335 cm/h for 39.4 kbp pDNA [26] and from 500 to 1000 cm/h for 6.9 kbp pDNA [20].…”

Amino acids–nucleotides biomolecular recognition: from biological occurrence to affinity chromatography

“…Similar to what has been described in the cellular mechanisms section about the influence of DNA structure in the protein-DNA interactions, in this case the topology of the pDNA also directly affected the interaction with amino acids and thus, the higher exposure degree of sc bases favoured the interaction with the His ligand. The influence of the plasmid topology in the interaction with His (Sousa et al, 2007b) was also described in a study where the plasmid retention was tested at different temperature conditions. It was verified that, with increase in the temperature, the plasmid suffered pre-denaturation conformational changes which promote the removal of negative superhelical turns in sc pDNA molecules.…”

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification

“…Histidine affinity chromatography is a promising alternative for the purification of pDNA from Escherichia coli (E. coli) cell lysate [10]. The underlying interaction mechanism is thought to involve hydrophobic, electrostatic and non-specific interactions of the nucleic acid bases and water mediated hydrogen bonds [11][12][13][14].…”