Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, w...

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Bibliographic Details
Published in:Biotechnology and bioengineering 2009-10, Vol.104 (3), p.505-515
Main Authors: Carnes, Aaron E, Hodgson, Clague P, Luke, Jeremy M, Vincent, Justin M, Williams, James A
Format: Article
Language:English
Subjects:
autolysis
Bacteriolysis
Biological and medical sciences
Biotechnology
Biotechnology - methods
Cells
Deoxyribonucleic acid
DNA
DNA - biosynthesis
DNA - isolation & purification
DNA vaccine
E coli
Endopeptidases - genetics
Endopeptidases - metabolism
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Fermentation
Fundamental and applied biological sciences. Psychology
Gene expression
gene therapy
lysis
Methods. Procedures. Technologies
Microbial engineering. Fermentation and microbial culture technology
plasmid
Plasmids - biosynthesis
Plasmids - isolation & purification
Selection, Genetic
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary:DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage λR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts. Biotechnol. Bioeng. 2009; 104: 505-515
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.22415