Negative pseudo-affinity chromatography for plasmid DNA purification using berenil as ligand

作者：Caramelo Nunes C; Gabriel M P; Almeida P; Marcos J C; Tomaz C T^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2014, 944: 39-42.

DOI：10.1016/j.jchromb.2013.10.043

• Berenil-Sepharose
• Negative pseudo-affinity chromatography
• Plasmid purification
• Small DNA ligands

The present study, reports the utilization of berenil as ligand in a negative pseudo-affinity chromatographic step to purify the plasmid pVAX1-LacZ from Escherichia coli clarified lysates. The chromatographic support was prepared by coupling berenil to epoxy-activated Sepharose and was qualitatively and quantitatively characterized using scanning electron microscopy, Fourier transformed infrared spectroscopy and elemental analysis. The clarified lysate was loaded onto the berenil-Sepharose support with 0.55 M of ammonium sulphate in the eluent, achieving the immediate elution of plasmid DNA. The impurities tightly bound to the support, were eluted after decreasing the salt concentration to 0 M. The overall process enabled the recovery of 87% of loaded plasmid DNA with a HPLC purity of >> 99% and according to FDA specifications. This method represents an alternative approach to the previous utilization of the same chromatographic pseudo-affinity support in a positive mode. It uses lower amounts of salt and one-step chromatographic procedure, resulting in smaller operating time and costs and representing an alternate procedure for plasmid DNA purification.

• 出版日期2014-1-1