Analytical Biochemistry 343 (2005) 183–185 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.03.011 ANALYTICAL BIOCHEMISTRY Notes & Tips Separation of supercoiled and open circular plasmid DNA isoforms by chromatography with a histidine–agarose support F. Sousa a , C.T. Tomaz a , D.M.F. Prazeres b , J.A. Queiroz a,¤ a Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal b Centro de Engenharia Biológica e Química, Instituto Superior Técnico, 1049-001 Lisboa, Portugal Received 10 February 2005 Available online 25 March 2005 The recent developments in molecular therapies, such as nonviral gene therapy and DNA vaccination, have fos- tered the development of eYcient plasmid DNA (pDNA) 1 puriWcation processes [1,2]. The separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large-scale puriWcation of pDNA vectors intended for therapeutic use. Furthermore, isoform separation, identiWcation, and quantitation are also crucial to monitor manufacturing and to control pDNA quality during pro- cessing and in the Wnal formulations [1]. Plasmids are usually produced in an Escherichia coli host by fermentation and are puriWed by a sequence of operations [3]. Although E. coli produces mainly the more compact sc pDNA isoform [3], oc, linear, and dena- tured pDNA isoforms are usually present due to confor- mational changes that occur within the bacterial host or during processing of biomass [4]. For instance, an intact and undamaged form of sc pDNA may give rise to oc or linear isoforms by random cleavage (e.g., enzymatic, chemical) of one or two opposing strands, respectively [5]. Furthermore, irreversibly denatured covalently closed pDNA isoforms are typically generated during alkaline lysis if the pH is not kept below 12.5 [3]. Whatever the case, the resulting isoforms are likely to be less eYcient in transferring gene expression if cleavage or strand melting disrupts promoter or gene coding regions [4]. For this reason, regulatory agencies specify that more than 90% of pDNA in a therapeutic product must be in the sc isoform [4]. The required separation of sc and oc pDNA isoforms has been accomplished by anion exchange [3,6] and hydrophobic interaction (HIC) [5,7] chromatography. HIC has also been used to separate native (oc + sc) pDNA from denatured pDNA isoforms [8]. In the HIC separations, the retention pattern of the diVerent pDNA species (sc, oc, or denatured) has been related to a diVer- ent degree of exposure of hydrophobic bases [8]. Histidine–base interactions have been recognized at the atomic level in several protein–DNA structures [9,10]. Building on those observations, this study explores histi- dine–base recognition as a means to separate pDNA iso- forms by pseudo-aYnity chromatography. Although histidine-based supports have been used for protein puri- Wcation [11], their application to DNA separation as described below is novel. The agarose gel used here com- bines the mild hydrophobic characteristics of an epoxy spacer arm [12] with a pseudo-aYnity histidine ligand. The 6.05-kbp plasmid pVAX1–LacZ (Invitrogen, Carlsbad, CA, USA) used in the experiments was pro- duced by a cell culture of E. coli DH5. Growth was car- ried out at 37 °C in a bioreactor with 1.5 L of TerriWc Broth medium (20 g/L tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 0.017 M KH 2 PO 4 , 0.072 M K 2 HPO 4 ) supplemented with 30 g/ml of kanamycin. The dis- solved oxygen concentration was kept at 30%, and growth was suspended at the late log phase (OD 600 t 20). Cells were recovered by centrifugation and were stored at ¡20 °C. Plasmid DNA was puriWed using the Qiagen (Hilden, Germany) plasmid midi kit according to the manufacturer’s instructions. The protocol is based on a modiWed alkaline lysis procedure. Following lysis, bind- ing of pDNA to the Qiagen anion exchange resin is pro- moted under appropriate low-salt and pH conditions. * Corresponding author. Fax: +351 275 319 883. E-mail address: jqueiroz@ubi.pt (J.A. Queiroz). 1 Abbreviations used: pDNA, plasmid DNA; sc, supercoiled; oc, open circular; HIC, hydrophobic interaction; FPLC, fast protein liquid chromatography.