Method for producing a recombinant protein on a manufacturing scale

A method for plasmid-free production of a heterologous protein of interest on a manufacturing scale, comprising the steps of

• a) cultivating bacterial expression host cells in a culture medium, said cultivating comprising a mode that employs adding a feeding medium, wherein said mode is selected from fed-batch mode, semi-continuous mode, and continuous mode,
• wherein
• (i) one or two copies of a DNA construct are integrated in the chromosome of the host cells, and wherein said DNA construct comprises one DNA sequence encoding the heterologous protein of interest under the control of a promoter that enables expression of the heterologous protein from the integrated one or two copies of the DNA construct,
• or
• (ii) one copy of a DNA construct is integrated in the chromosome of the host cells, and wherein said DNA construct comprises two copies of a DNA sequence encoding the heterologous protein of interest under the control of at least one promoter that enables expression of the heterologous protein from the integrated one copy of the DNA construct;
• wherein the DNA construct comprises a ribosome binding site, and optionally further comprises one or more components selected from sequences coding for antibiotic selection markers, prototrophic selection markers or fluorescent markers, genes coding for metabolic markers, and genes that improve protein expression, and flippase recognition target sites,
• and
• wherein the one or two copies of the DNA constructs are maintained in the chromosome of the cultivated expression host cells, and
• wherein the DNA sequence encoding the heterologous protein of interest is not present in a plasmid, and
• wherein cultivating takes place under conditions where the heterologous protein of interest is expressed;
• b) harvesting the heterologous protein of interest expressed by the expression host cells;
• and
• c) isolating and purifying the heterologous protein of interest on a manufacturing scale.