Plasmid DNA preparations and methods for producing same
Inventors
Blattner, Frederick R. • Campbell, John Walter • Plunkett, Guy
Assignees
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Publication Number
US-7935505-B2
Publication Date
2011-05-03
Expiration Date
Abstract
The various embodiments of the present invention relate generally to plasmid DNA preparations and to methods for producing and using such preparations.
Core Innovation
The invention provides an Escherichia coli multiple deletion strain that lacks a functional msbB gene and one or more genes involved in production of enteric common antigen, selected from genes located on the E. coli strain K12 MG1655 chromosome between nucleotide 3965939 and 3980295. Plasmid DNA preparations are produced using the defined multiple deletion strain.
The plasmid DNA preparations show reduced endotoxin compared with non-multiple-deletion controls, including embodiments with endotoxin levels down to very low EU/mg prior to endotoxin-removal-agent treatment. Endotoxin levels are evaluated by LAL assays.
The document further describes that endotoxin reduction is associated with improved functional behavior of the plasmid DNA in cell-based contexts. Example results include reduced cytotoxicity and improved COS1 cell transfection efficiency, with particular improvement described when ECA cluster deletion is combined with an msbB mutation.
Claims Coverage
The independent claims define two inventive features: a specific E. coli multiple deletion strain and a method for producing plasmid DNA with reduced endotoxin using that strain. The claims focus on deleting msbB and selected enteric common antigen genes from a defined MG1655 chromosome interval, and on producing plasmid DNA having a specified endotoxin level prior to purification using an endotoxin removal agent.
Multiple deletion strain lacking msbB and selected ECA genes from MG1655 interval
An E. coli multiple deletion strain which lacks a functional msbB gene and one or more genes involved in production of enteric common antigen, selected from the genes located on the E. coli strain K12 MG1655 chromosome between nucleotide 3965939 and 3980295.
Method producing plasmid DNA with reduced endotoxin using the defined multiple deletion strain
A method that produces plasmid DNA with reduced endotoxin levels by using the multiple deletion strain as defined in claim 1 to grow bacteria, isolate plasmid DNA from the lysate, and purify the isolated plasmid DNA.
Plasmid DNA with low endotoxin before purification with an endotoxin removal agent
The method includes using plasmid DNA having less than 200 EU/mg endotoxin before purifying the plasmid DNA with an endotoxin removal agent.
Overall, the claims coverage centers on an E. coli multiple deletion strain defined by msbB loss together with loss of enteric common antigen genes from a specified MG1655 chromosomal interval, and on plasmid DNA production using that strain to obtain plasmid DNA with reduced endotoxin, including a stated endotoxin threshold (<200 EU/mg) before purification with an endotoxin removal agent.
Stated Advantages
Reduced endotoxin levels in plasmid DNA preparations versus non-multiple-deletion controls.
Improved COS1 cell transfection efficiency associated with reduced endotoxin.
Reduced cytotoxicity in COS1 cells associated with reduced endotoxin.
Documented Applications
Use of the described plasmid DNA preparations in COS1 monkey kidney cells for transfection, where improved transfection efficiency and reduced cytotoxicity are described.
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