title: Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A.
abstract: Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH ( approximately 12-12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18DeltalacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.
authors.affiliation: The Advanced Centre For Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
authors.forename: G D
authors.initials: GD
authors.lastname: Cooke
authors.forename: R M
authors.initials: RM
authors.lastname: Cranenburgh
authors.forename: J A
authors.initials: JA
authors.lastname: Hanak
authors.forename: P
authors.initials: P
authors.lastname: Dunnill
authors.forename: D R
authors.initials: DR
authors.lastname: Thatcher
authors.forename: J M
authors.initials: JM
authors.lastname: Ward
mesh_terms: D000818:Animals; D001709:Biotechnology; D002417:Cattle; D002455:Cell Division; D004269:DNA, Bacterial / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification; D004340:Drug Contamination; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000235:genetics* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012329:RNA, Bacterial; D012259:Ribonuclease, Pancreatic