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Plasmid DNA production combining antibiotic‐free selection, inducible high yield fermentation, and novel autolytic purification
Author(s) -
Carnes Aaron E.,
Hodgson Clague P.,
Luke Jeremy M.,
Vincent Justin M.,
Williams James A.
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22415
Subject(s) - lysin , plasmid preparation , plasmid , lysis , biology , dna , escherichia coli , autolysis (biology) , peptidoglycan , downstream processing , alkaline lysis , fermentation , microbiology and biotechnology , cytoplasm , bacteria , genomic dna , biochemistry , cell wall , gene , bacteriophage , enzyme , dna vaccination , genetics , pbr322
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 © 2009 Wiley Periodicals, Inc.