NDLI: Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines

Content Provider	Springer Nature Link
Author	Prather, Kristala L. Jones Edmonds, M. Celina Herod, Jerrell W.
Copyright Year	2006
Abstract	Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.
Starting Page	815
Ending Page	826
Page Count	12
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	73
Issue Number	4
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2006-08-29
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	DNA vaccine IS1 transposition Q-PCR RFLP Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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