## 3 Results and Discussion  
### Preparative Chromatography  
The clarification procedure performed after cell lysis enables the preparation of an extract enriched in sc pDNA and simultaneously promotes the reduction of the impurities content, although it is not possible to fully eliminate species such as RNA, gDNA, endotoxins, or host proteins. Therefore, affinity chromatography using amino acids as ligands has been extensively used by our research group to obtain a final pDNA sample fulfilling the requirements of regulatory agencies [22; 23; 24]. Of all the amino acids studied, arginine and histidine have shown to be the most promising chromatographic ligands, allowing the purification of different plasmids with high recovery yield and purity degree [24; 25]. In the present study, the selection of these amino acids was also performed because they present completely different chromatographic behaviors for the isolation of sc pDNA. In fact, through the literature, it is possible to conclude that although multiple interactions occur between the nucleic acids and these amino acids, the main interactions established with arginine are ionic interactions, while the histidine ligand mainly promotes hydrophobic interactions. This information will influence the choice of the type of salt and gradient to be applied but other parameters such as temperature or pH are crucial to establish the ideal conditions for sc pDNA purification while ensuring its stability. In this way, the main goal of this study is to understand how different chromatographic approaches influence the bioactivity of the recovered sc pDNA. Initially, three different supports were applied for the isolation and purification of sc p53 encoding pDNA, according to the optimized strategies described in Figure 2.  
It is possible to observe the difference of the binding behavior represented on the three chromatographic profiles. The histidine-agarose support was used to purify the sc pDNA from the clarified lysate sample by a decreasing stepwise gradient of ammonium sulfate (Figure 2A). The open circular (oc) pDNA isoform was eluted in peak 1 with 2.3 M (NH4)2 SO4 in 10 mM Tris-HCl buffer (pH 8.0), as it can be visualized in lane 1 of the agarose gel electrophoresis (Figure 2A). The sc pDNA  
Figure 2: Chromatographic profiles and respective agarose gel electrophoresis of the p53-pDNA purification from an _E. coli_ lysate using different chromatographic supports. **(A)**—Histidine–agarose matrix: the elution was performed by a decreasing stepwise gradient of 2.3 M (NH4)2 SO4 in 10 mM Tris–HCl, pH 8.0, then 1.7 M (NH4)2 SO4 in the same buffer, and finally 10 mM Tris–HCl, pH 8.0; **(B)**—Arginine–macroporous support: using an increasing stepwise gradient of 124 mM NaCl in 10 mM Tris–HCl, pH 8.0, then 500 mM of NaCl in the same buffer, and finally 2 M NaCl in Tris–HCl, pH 8.0; **(C)**—Histidine–monolith: in this support it was used 3 M (NH4)2 SO4 in 10 mM citrate–EDTA, pH 5.0, then 2.52 M (NH4)2 SO4 in the same buffer, and finally 10 mM citrate–EDTA, pH 5.0. In the agarose gel electrophoresis, lane L represents the lysate, and lanes 1, 2, and 3 represent the first, second, and third peaks of the respective chromatogram. MP*: Mobile phase; SP**: Stationary phase; RT: Room temperature. isoform was eluted in the second peak, at 1.7 M (NH4)2 SO4 (lane 2 of agarose electrophoresis--Figure 1A), and finally, the third peak corresponds to the elution of more strongly bound molecules, recovered with 10 mM Tris-HCl buffer (pH 8.0), as observed in lane 3 of the agarose gel electrophoresis, by the presence of RNA and small amounts of other molecular species (Figure 2A). The dominant interactions responsible for the nucleic acid binding behavior on this column are the hydrophobic ones since the imidazole ring is able to promote interactions with nitrogenous bases of nucleic acids, due to the influence of high ionic strength on the mobile phase.