Premium
Stability studies with different vector backbones utilizing the T7 expression system in Escherichia coli
Author(s) -
Walia Rupali,
Deb Jahar Kanti,
Mukherjee Krishna Jyoti
Publication year - 2008
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.1885
Subject(s) - escherichia coli , plasmid , recombinant dna , expression vector , biology , vector (molecular biology) , mutant , gene , microbiology and biotechnology , chemistry , biochemistry
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