Vector Design

The expression vector is basically divided into prokaryotic and eukaryotic elements. For high-copy number replication in E. coli an origin of replication and a selectable marker are required. To achieve high-titer TGE, the plasmid needs an expression cassette for gene expression in mammalian cells regulated by a strong promoter. The human cytomegalovirus immediate early 1 (hCMV-IE1) promoter is one of the most commonly used promoters for recombinant protein expression in mammalian cells. Further basic elements are the multiple cloning site (MCS) and the polyA terminator sequence. For detailed information, take a closer look at the schematic figure of our plasmid pINV. pINV is a very small plasmid and lacks popular genetic elements as mentioned in the cell line section. Our results indicate that productivity in TGE is highly correlated to the quantity of plasmid DNA which reaches the nucleus. Since transfection efficiency of your plasmid is limited by the cytotoxicity of the transfection reagent used, it is reasonable to minimize the vector backbone to increase the molarity within the nucleus. Therefore pINV consists of a miniscule replication origin pUC19ori, a small marker mediating antibiotic resistance for amplification in E. coli and a minimized human cytomegalovirus promoter for strong gene expression. Our high-titer TGE vector also contains an optimal signal peptide included into the coding sequence of the target protein to ensure full secretion and maximum yield of the expressed protein in downstream processing. An optimized codon usage, a prefixed Kozak sequence as efficient protein translation initiation site and different tags complement our vector system.