Specific recognition of supercoiled plasmid DNA in arginine affinity chromatography

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

“…In this study, it was also verified that His interacts preferentially with the guanine and adenine bases (Sousa et al, 2009b), as described at atomic level (Luscombe et al, 2001;Hoffman et al, 2004). The similar studies performed using Arg (Sousa et al, 2008b) and Lys (Sousa et al, 2009a) chromatography to purify pDNA, also proved the presence of a specific interaction with plasmid molecules, and a particular recognition of sc isoform. Considering the previous discussion resulting from the analysis of the protein-DNA interactions at atomic level, the interaction occurring between pDNA and Arg support was supposed to be rather complex, because apart from the stability promoted by the interaction with pDNA backbone, the ability of the Arg-agarose to distinguish and differentially interact with both isoforms suggested a specific recognition of the sc pDNA isoform.…”

“…The screening of the ability of different amino acids such as Leu, Gln, Asp, His, Arg and Lys to isolate sc pDNA showed that this biomolecule presents a selective interaction with His (Sousa et al, 2005), Arg (Sousa et al, 2008b) and Lys (Sousa et al, 2009a). The application of His as ligand revealed a different recognition for the different nucleic acids, being the structure and topology of these biomolecules one of the major issues influencing the interaction.…”

Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification

“…Amino acids ligands were used in affinity chromatography of proteins (Kanoun et al, 1986;Wu et al, 1992) (Jahreis et al, 2001), oligosaccharides (Delattre et al, 2005) and endotoxins (Wei et al, 2007), however, very recently was asserted for the first time their application for pDNA purification. In these latest works we have described the total removal of E. coli impurities using a histidine-agarose matrix (Sousa et al, 2006) and the baseline separation of sc and oc isoforms either using histidine (Sousa et al, 2005) or arginine as chromatographic ligands (Sousa et al, 2008). The fact that considerably lower salt concentration must be used to isolate sc isoform with arginine-based matrix also revealed an important process advantage (Sousa et al, 2008), suggesting the feasibility of exploiting affinity interactions of amino acids with nucleic acids to develop an interesting bioseparation methodology.…”

“…In these latest works we have described the total removal of E. coli impurities using a histidine-agarose matrix (Sousa et al, 2006) and the baseline separation of sc and oc isoforms either using histidine (Sousa et al, 2005) or arginine as chromatographic ligands (Sousa et al, 2008). The fact that considerably lower salt concentration must be used to isolate sc isoform with arginine-based matrix also revealed an important process advantage (Sousa et al, 2008), suggesting the feasibility of exploiting affinity interactions of amino acids with nucleic acids to develop an interesting bioseparation methodology. However, to achieve higher elution efficiency in affinity chromatography, other conditions than the ionic strength need to be tested and controlled to improve the purification strategies.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…The screening of the ability of histidine and arginine to isolate sc pDNA has shown that both amino acids ligands promote a specific interaction with pDNA. Extending beyond the possibility of histidine-and arginine-based affinity matrixes to isolate the major plasmid isoforms (oc and sc pDNA) Sousa et al 2008a), the applicability of these matrices for the efficiently purification of sc pDNA from host impurities present in a clarified E. coli lysate (Sousa et al 2006;Sousa et al 2009c) has been further demonstrated by those researchers. In the case of histidinechromatography, the application of an ammonium sulphate gradient appears to allow the specific recognition of sc pDNA by the histidine ligands, whereas no interaction has been found with oc pDNA and gDNA.…”