Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A

• Published in: Journal of biotechnology Vol. 85; no. 3; pp. 297 - 304
• Main Authors: Cooke, G.D., Cranenburgh, R.M., Hanak, J.A.J., Dunnill, P., Thatcher, D.R., Ward, J.M.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 23.02.2001; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cattle; Cell Division; Chromosomal integration; DNA; DNA, Bacterial - isolation & purification; DNA, Recombinant - genetics; DNA, Recombinant - isolation & purification; Drug Contamination; Enzyme kinetics; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene therapy; Genetic Therapy; Methods. Procedures. Technologies; Others; Periplasm; Plasmids - genetics; Plasmids - isolation & purification; Purification; ribonuclease A; Ribonuclease, Pancreatic - genetics; Ribonuclease, Pancreatic - metabolism; RNA; RNA, Bacterial - isolation & purification; RNase; Various methods and equipments; aa, amino acid(s); EDTA, ethylenediaminetetraacetic acid; kb, kilobase(s) or 1000 bp; CJD, Creutzfeldt–Jakob disease; bp, base pair(s); A, absorbance; TE, 10 mM Tris–HCl, 1 mM EDTA buffer; IPTG, iospropyl β- d-thiogalactopyranoside; λ, Bacteriophage lambda; cDNA, DNA complementary to RNA; RNaseA, bovine pancreatic ribonuclease; SDS, sodium dodecyl sulphate; Chromosomal integration; RNase; WHO, World Health Organisation; OD, optical density; FDA, Food and Drug Administration; p, plasmid; TBE, tris–borate EDTA buffer; EMEA, European Agency for the Evaluation of Medicinal Products; TSE, transmissible spongiform encephalopathies; RNase, ribonuclease; dNTP, deoxyribonucleoside triphosphate; EtdBr, ethidium bromide; Periplasm; P, promoter; Gene therapy; Recombinant microorganism; Purification; RNA; Enzyme; Escherichia coli; Esterases; Plasmid; DNA; Bacteria; Hydrolases; Pancreatic ribonuclease; Application; Biological contamination; Enterobacteriaceae
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(00)00378-3

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 (∼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 pUC18Δ lacO, 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.