Patent ID: 9683252
Date: 2017-06-20
CPC Classifications: C12N,C12P

Claim:
1. A method for plasmid-free production of a heterologous protein of interest on a manufacturing scale, consisting of the steps of a) cultivating expression host cells that are wherein one or two copies of a DNA construct are integrated in the chromosome of the host cells, and wherein said DNA construct consists of a 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 said DNA construct, a ribosome binding site, two terminally flanking regions homologous to a genomic region of the host cells that enable homologous recombination, and optionally one or more of the components selected from gene 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 expression host cells contain a T7 RNA polymerase gene in the chromosome and wherein said promoter is a T7 promoter, wherein the one or two copies of said 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; b) harvesting the heterologous protein of interest expressed by the expression host cells; and c) isolating and purifying the heterologous protein of interest.