Purification of minicircles by combined enzymatic modification of miniplasmid topology and multimodal chromatography
March 5-10, 2017
Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by multimodal chromatography.
Please click Additional Files below to see the full abstract.
Duarte Miguel Prazeres, Ana Rita Silva-Santos, Cláudia Alves, Michaela Šimčiková, Gabriel Monteiro, and Ana Margarida Azevedo, "Purification of minicircles by combined enzymatic modification of miniplasmid topology and multimodal chromatography" in "Separations Technology IX: New Frontiers in Media, Techniques, and Technologies", Kamalesh K. Sirkar, New Jersey Institute of Technology, USA Steven M. Crame, Rensselaer Polytechnic Institute, USA João G. Crespo, LAQV-Requimte, FCT-Universidade Nova de Lisboa, Caparica, Portugal Marco Mazzotti, ETH Zurich, Switzerland Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/separations_technology_ix/45