Method for producing high yield attenuated Salmonella strains
Inventors
Lubenau, Heinz • Siede, Holger • Janssen, Renate • Springer, Marco
Assignees
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Publication Number
US-9493738-B2
Publication Date
2016-11-15
Expiration Date
Abstract
This invention relates to a novel method for growing attenuated mutant Salmonella typhi strains lacking galactose epimerase activity and harboring a recombinant DNA molecule. The method comprises the step of culturing said Salmonella typhi strain without adding glucose to the medium during the fermentation with a starting glucose amount that is depleted before reaching the stationary phase. The invention further relates to attenuated mutant Salmonella typhi strains obtainable by said method and to an attenuated mutant Salmonella typhi strain harboring a recombinant DNA molecule encoding a VEGF receptor protein for use as a vaccine.
Core Innovation
The invention relates to a method for growing an attenuated mutant strain of Salmonella typhi lacking galactose epimerase activity, wherein the strain comprises at least one copy of a recombinant DNA molecule comprising a eukaryotic expression cassette. The method includes culturing the strain in a buffered medium comprising peptone at approximately neutral starting pH value at fermentation scale, using a medium volume of at least about 10 l.
During fermentation, no glucose is added to the medium, and the amount of glucose in the medium during the fermentation is adjusted such that the amount of glucose is reduced to zero before reaching the stationary phase. The buffered peptone medium is described as including peptone and, in examples, can include non-animal TSB, with approximately neutral starting pH, including ranges such as about 6 to about 8 and about 6.5 to about 7.5.
The invention is also tied to the transformed organism, including examples of attenuated Salmonella typhi such as Ty21a transformed with recombinant DNA encoding a VEGF receptor, notably human VEGFR-2, for DNA vaccine use. In exemplified recombinant DNA embodiments, a eukaryotic expression cassette expresses human VEGFR-2 under a CMV promoter, and the recombinant DNA molecule can include a kanamycin resistance gene and a pMB1 origin of replication, with VEGFR-2 sequences identified by SEQ ID NO 1 and SEQ ID NO 2.
Claims Coverage
The document contains one independent claim covering fermentation-scale growth of a galactose-epimerase-deficient attenuated Salmonella typhi mutant carrying recombinant DNA with a eukaryotic expression cassette, with glucose management and neutral buffered peptone conditions as central inventive limitations.
Fermentation-scale growth in buffered peptone at approximately neutral starting pH
Culturing an attenuated mutant strain of Salmonella typhi lacking galactose epimerase activity and comprising at least one copy of a recombinant DNA molecule comprising a eukaryotic expression cassette, in a buffered medium comprising peptone at approximately neutral starting pH value at fermentation scale.
Glucose reduction to zero before stationary phase without glucose addition
Culturing where no glucose is added to the medium during the fermentation, wherein the amount of glucose in the medium during the fermentation is adjusted such that the amount of glucose is reduced to zero before reaching the stationary phase.
Fermentation volume of at least about 10 l
Performing the culturing using a volume of the medium that is at least about 10 l.
Across the independent claim, the main coverage is directed to fermentation-scale culturing of an attenuated galactose-epimerase-deficient Salmonella typhi mutant carrying recombinant DNA with a eukaryotic expression cassette, using buffered peptone at approximately neutral starting pH, while managing glucose such that it is reduced to zero before the stationary phase, and using a medium volume of at least about 10 l.
Stated Advantages
Provides higher OD600/cell density/CFU than glucose-sustained cultivations, as supported by quantitative comparisons described in the provided content.
Documented Applications
DNA vaccine use using attenuated Salmonella typhi transformed with recombinant DNA encoding a VEGF receptor, notably human VEGFR-2.
Oral cancer vaccine delivery via attenuated Salmonella, as described in the provided content.
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