title: Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy.
abstract: Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA. Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alkaline lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA. Although it is possible to minimize plasmid degradation by decreasing temperature and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams. The kinetics of nucleic acid degradation showed that, by controlling the incubation at 37 degrees C, it was possible to degrade RNA selectively, while maintaining plasmid integrity. A reduction of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liquid chromatography (HPLC). This strategy is simple and straightforward, and the increase in processing time and the associated plasmid loss (9%) are fully justified by the purity increase. Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addition of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies.
authors.affiliation: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1000 Lisboa, Portugal.
authors.forename: G A
authors.initials: GA
authors.lastname: Monteiro
authors.forename: G N
authors.initials: GN
authors.lastname: Ferreira
authors.forename: J M
authors.initials: JM
authors.lastname: Cabral
authors.forename: D M
authors.initials: DM
authors.lastname: Prazeres
mesh_terms: D001709:Biotechnology; D004269:DNA, Bacterial / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D010957:Plasmids / Q000302:isolation & purification* / Q000378:metabolism / Q000627:therapeutic use; D012329:RNA, Bacterial / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D003851:Deoxyribonucleases; D012260:Ribonucleases