Stability studies with different vector backbones utilizing the T7 expression system in Escherichia coli

BACKGROUND: The T7 system is used ubiquitously for high-level expression of recombinant proteins in lab scale cultures of Escherichia coli. However, its functional stability during scale-up is critical for it to be useful in industrial scale fermentations. RESULTS:E. coli DH5α cells containing the dual plasmid heat inducible T7 system were tested for sustained expression of interferon gamma for 60 generations. The pRSET-IFNγ recombinant lost its ability to express h-IFNγ after 30 generations even though the plasmid containing the unaltered sequence of the gamma interferon gene was retained. These non-expressing mutants had a higher specific growth rate than the original recombinant cells. In contrast, pCR2-IFNγ recombinant had a higher specific growth rate under uninduced conditions and was able to retain its expression capacity even in the absence of selection pressure. To demonstrate the usefulness of this construct a high cell density fed batch culture was run without ampicillin and high-level expression was obtained. CONCLUSIONS: The pCR2 vector backbone is a useful addition to the repertoire of E. coli plasmids, particularly for scaling up high-level expression systems in the absence of selection pressure. Copyright © 2008 Society of Chemical Industry