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pET SYSTEM

pET SYSTEM

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Published by: navyeinstein1991 on Apr 16, 2011
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08/20/2011

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As first described by Grossman et al. (1998), low basal expression levels in the pET
system can be maintained by supplementing the medium with glucose. As cultures reach
stationary phase, any available glucose is consumed first and an alternative carbon source
such as glycerol is then utilized. Metabolism of the alternate carbon source causes cyclic
AMP (cAMP) levels to increase, stimulating transcription from the lacUV5 promoter and
subsequent expression of T7 RNA polymerase in λDE3 lysogens. In contrast to the wild-
type lac promoter, the lacUV5 promoter is not as sensitive to cAMP stimulation (Eron et
al., 1971; Fried et al.,1984). However, it has been demonstrated that sufficient stimulation
occurs to elevate T7 RNA polymerase levels, and consequently, T7 promoter regulated
target gene expression (Grossman et al., 1998; Kelley, 1995; Novy et al., 2001; Pan et al.,
2000). A significant decrease in basal transcription from the lacUV5 promoter is observed
when standard medium is supplemented with glucose in cultures grown to stationary
phase (Grossman et al., 1998; Novy et al., 2001; Pan et al., 2000).

Minimizing basal expression is particularly important for pET vector expression when
hosts that do not carry the pLysS plasmid are allowed to grow to stationary phase
(16 h; overnight cultures) and when the target gene is toxic (Grossman et al., 1998; Novy
et al., 2001). Without the T7 lysozyme from the pLysS plasmid, basal expression levels are
elevated in cultures grown to stationary phase. If the gene is toxic, the addition of 0.5–1%
glucose to both liquid medium and agar plates may be necessary to maintain plasmid
stability. Hosts containing pLysS may express an elevated level of lysozyme in cultures
grown to stationary phase such that induced levels of the target protein are lowered. This
is likely because the CAT gene promoter is also sensitive to stimulation by cAMP in the
absence of glucose and is upstream of the T7 lysozyme gene in pLysS (Novy et al., 2001).

The addition of glucose is neither necessary nor recommended during the cloning
steps in non-expression hosts. Although growing cultures to stationary phase is not
recommended, glucose provides another method to maintain the lowest basal levels of
target protein in λDE3 lysogenic expression hosts used in the pET System and prevents
overproduction of T7 lysozyme.

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