Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase.
BACKGROUND: Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure. RESULTS: We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(lambdaDE3) reproducibly restored high level protein production. CONCLUSIONS: Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate.
Vethanayagam
Joe Gg
JG
Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, USA. aflower@medicine.nodak.edu.
Flower
Ann M
AM