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A process is described that uses a single histidine\u2013agarose chromatography step to purify sc pDNA from other isoforms and <jats:italic>Escherichia coli<\/jats:italic> impurities present in a clarified lysate. The histidine\u2013agarose support combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo\u2010affinity histidine ligand. The 6 kb DNA vaccine backbone pVAX1\u2010LacZ was used as a model target. Following loading at high salt [2.3 M (NH<jats:sub>4<\/jats:sub>)<jats:sub>2<\/jats:sub>SO<jats:sub>4<\/jats:sub>], the different species were eluted by a series of reverse salt step gradients (2.0, 1.5 and 0 M (NH<jats:sub>4<\/jats:sub>)<jats:sub>2<\/jats:sub>SO<jats:sub>4<\/jats:sub>). Open circular pDNA and gDNA was eluted at 2.3 M, sc pDNA was isolated as a single peak at 2.0 M and RNA was eluted at 1.5 M (NH<jats:sub>4<\/jats:sub>)<jats:sub>2<\/jats:sub>SO<jats:sub>4<\/jats:sub> and lower. The underlying mechanism is thought to involve not only hydrophobic interactions between the support and pDNA molecules, but also non\u2010specific biorecognition of nucleic acid bases by the histidine ligand. Control analysis showed that the isolated sc pDNA conforms to specifications in terms of gDNA (3.4 ng\/\u03bcg of pDNA), endotoxins (0.02 endotoxin unit\/\u03bcg of pDNA), RNA and proteins (undetectable) and pDNA homogeneity (\u223c100% sc). Furthermore, the transfection efficiency of Chinese\u2010hamster ovary cells (50%) was significantly higher when compared with the efficiency (25%) of a pDNA control. The present study confirms the possibility of using a single histidine\u2013agarose chromatography step to purify sc pDNA from other isoforms and host contaminants present in a clarified <jats:italic>E. coli<\/jats:italic> lysate.<\/jats:p>","DOI":"10.1042\/ba20060082","type":"journal-article","created":{"date-parts":[[2006,10,19]],"date-time":"2006-10-19T10:41:31Z","timestamp":1161254491000},"page":"131-140","source":"Crossref","is-referenced-by-count":68,"title":["Selective purification of supercoiled plasmid DNA from clarified cell lysates with a single histidine\u2013agarose chromatography step"],"prefix":"10.1002","volume":"45","author":[{"given":"Fani","family":"Sousa","sequence":"first","affiliation":[]},{"given":"Sind\u00e9lia","family":"Freitas","sequence":"additional","affiliation":[]},{"given":"Adriano R.","family":"Azzoni","sequence":"additional","affiliation":[]},{"given":"Duarte Miguel F.","family":"Prazeres","sequence":"additional","affiliation":[]},{"given":"Jo\u00e3o","family":"Queiroz","sequence":"additional","affiliation":[]}],"member":"311","published-online":{"date-parts":[[2010,12,23]]},"reference":[{"key":"e_1_2_8_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0905-1059"},{"key":"e_1_2_8_3_2","doi-asserted-by":"publisher","DOI":"10.1002\/jgm.511"},{"key":"e_1_2_8_4_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.13.7507"},{"key":"e_1_2_8_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2004.09.050"},{"key":"e_1_2_8_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2005.05.003"},{"key":"e_1_2_8_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0167-5699(97)01201-2"},{"key":"e_1_2_8_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9673(97)01254-5"},{"key":"e_1_2_8_9_2","doi-asserted-by":"publisher","DOI":"10.1023\/A:1016361721316"},{"key":"e_1_2_8_10_2","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0290(20000605)68:5<576::AID-BIT13>3.0.CO;2-5"},{"key":"e_1_2_8_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1387-2656(01)07031-4"},{"key":"e_1_2_8_12_2","doi-asserted-by":"crossref","first-page":"92","DOI":"10.2144\/96211st03","volume":"21","author":"Davis H. 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