ABSTRACT
We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.
Subject(s)
Aldehyde-Lyases/biosynthesis , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/physiology , Operator Regions, Genetic , Plasmids , Repressor Proteins/genetics , Aldehyde-Lyases/analysis , Animals , Escherichia coli/enzymology , Fermentation , Gene Expression Regulation, Bacterial/genetics , Industrial Microbiology/methods , Plasmids/genetics , Plasmids/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Repressor Proteins/metabolismABSTRACT
Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.
Subject(s)
Drug Resistance/genetics , Plague Vaccine/immunology , Plasmids , Salmonella typhimurium/genetics , Vaccines, Synthetic/immunology , Animals , Female , Mice , Mice, Inbred BALB C , Salmonella typhimurium/immunologyABSTRACT
The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model.
