Methods for production of recombinant plasmids

A culture system for stable, high-level production of recombinant plasmids comprising genetically engineered bacterial cells and recombinant plasmids,

• wherein the bacterial cell chromosome is irreversibly modified so as to render the cell incapable of producing an essential metabolite and also incapable of the uptake of said metabolite from a culture medium,
• wherein the essential metabolite is lysine, and
• wherein the inability to synthesize lysine is effected by a mutation in the .sub.lysA gene and the inability to take up lysine from the medium is effected by a disruption in the .sub.lysp gene,
• wherein the recombinant plasmid includes genetic material which restores either the ability to synthesize said metabolite or the ability to take up the metabolite from the medium or both and which optionally further includes foreign DNA operatively linked to eukaryotic control sequences whereby said foreign DNA is not expressed in said bacterial cells,
• wherein said genetic material has no functional or structural equivalent in animal cells;
• and
• wherein any protein expressed by said genetic material is incapable of acting upon any animal cell component;
• and
• wherein any protein expressed by said genetic material or product resulting from the presence of said protein is not secreted by or toxic to said bacterial cells.

A culture system for stable high-level production of recombinant plasmids comprising genetically engineered bacterial cells and recombinant plasmids, wherein the bacterial cell chromosome is irreversibly altered and the bacterial cells are propagated under conditions such that the viability of the bacterial cells is dependent on the recombinant plasmid, and wherein the plasmid includes genetic material which functionally complements the chromosomal alteration,

• wherein the chromosome is altered so as to result in the inability of said bacterial cells to synthesize an enzyme that catalyzes a step in the biosynthesis of a cell wall;
• wherein said enzyme catalyzes a reaction selected from the group consisting of the addition of L-alanine to a cell wall polysaccharide (L-Ala-adding enzyme);
• the addition of D-glutamic acid to the L-alanine attached to the polysaccharide chain (.sub.murD);
• the addition of dap to the L-alanine/D-glutamic acid dipeptide (.sub.murE);
• and the addition of D-alanine to the L-alanine/D-glutamic acid/dap tripeptide of the bacterial cell wall (.sub.murF);
• wherein said genetic material comprises a nucleotide sequence encoding said enzyme;
• wherein said genetic material has no functional or structural equivalent in animal cells;
• wherein any protein expressed by said genetic material is incapable of acting upon any animal cell component;
• wherein any protein expressed by said genetic material or product resulting from the presence of said protein is not secreted by or toxic to said bacterial cells;
• and
• wherein the plasmid further includes foreign DNA operatively linked to eukaryotic control sequences whereby the foreign DNA is not expressed in said bacterial cells, but is expressed in animal cells upon delivery of said plasmid.