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

Abstract

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.

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

“…Moreover, for large-scale fermentation, plasmid selection using antibiotics can prove very costly [ 3 ] Drawbacks associated with antibiotic–based selection have therefore encouraged the development of various antibiotic marker-free selection approaches. Many such systems have been reported [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] but are not extensively adopted by the labs for a variety of reasons that, among other, include use of specialized strains and/or reagents.…”

Vector Insert-Targeted Integrative Antisense Expression System for Plasmid Stabilization

“…Improved copy number was observed under uninduced conditions (30°C) wherein only low levels of RNA were produced from the temperature inducible p R p L promoters and after temperature induction (42°C) of the p R p L promoters. The improved yield was not due to a nonspecific effect of pINT PR PL vector insertion since no yield difference with multiple plasmids was observed between DH5a and NTC3012, a DH5a derivative with a single copy of the pAH144-Lambda Repressor zwflambdaR plasmid integrated into the HK022 attachment site [16]. This vector is a protein expressing version of the pINT PR PL plasmid.…”

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

“…This approach is economically more feasible and easier to control than traditional methods of cell disruption (reviewed by Gao et al, 2013). Furthermore, phage endolysins have been used for DNA, RNA and protein release from Gram-positive bacteria such as Listeria (Loessner et al, 1995) or S. pyogenes (Köller et al, 2008) and for the development of auto-inducible or heat-inducible E. coli lysis system to facilitate protein expression (Zhang et al, 2009;Carnes et al, 2009).…”