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, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) 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.
Carnes
Aaron E
AE
Nature Technology Corporation, 4701 Innovation Drive, Lincoln, Nebraska 68521, USA. acarnes@natx.com
Hodgson
Clague P
CP
Luke
Jeremy M
JM
Vincent
Justin M
JM
Williams
James A
JA
D001433:Bacteriolysis; D001709:Biotechnology / Q000379:methods*; D004247:DNA / Q000096:biosynthesis* / Q000302:isolation & purification*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D010957:Plasmids / Q000096:biosynthesis* / Q000302:isolation & purification*; D012641:Selection, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D004247:DNA; D010450:Endopeptidases; C001143:endolysin