Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Vethanayagam, Joe G.; Flower, Ann M.

doi:10.1186/1475-2859-4-3

Cited by 47 publications

(18 citation statements)

References 11 publications

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“…Importantly, YidC was C-terminally fused to GFP since the GFP-moiety conveniently enabled to screen already on plate for IPTG-resistant BL21(DE3)-derivatives producing YidC-GFP in the cytoplasmic membrane33. This enabled us to rapidly filter out IPTG-resistant BL21(DE3) derivatives that somehow had accumulated mutations abolishing YidC-GFP production34. Using this approach one mutant, was isolated that was not only IPTG-resistant, but also fluorescent.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

Baumgarten

Schlegel

Wagner

et al. 2017

Sci Rep

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Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

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Section: Discussionmentioning

confidence: 99%

Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

Baumgarten

Schlegel

Wagner

et al. 2017

Sci Rep

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“…A recent report has shown that expression levels declined drastically in E. coli BL21 (DE3) cells containing a pET-based expression plasmid on repeated sub-culturing which could be traced to chromosomal mutations that diminished the level of functional T7 RNA polymerase. 9 A way to circumvent this problem would be to have multiple copies of the T7 RNA polymerase gene on a second plasmid as is the case with the heatinducible dual plasmid system. 10,11 We decided to investigate the stability of these dual plasmid systems by repeated sub-culturing to test their usefulness in an industrial setting.…”

Section: Introductionmentioning

confidence: 99%

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

Walia

Deb

Mukherjee

2008

J of Chemical Tech & Biotech

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“…However, the system is not without its problems, especially a gradual decrease in the expression levels of the target gene. After several (3 to 5) subcultures, the induced expression levels may be comparable to basal expression levels, and often the host cells do not express detectable levels of the target protein [5]. This results in a lower target protein yield.…”

Section: Introductionmentioning

confidence: 99%

“…Many attempts have been made to stabilize the expression levels of the T7 p/p system, including examination of plasmid stability by lowering the uninduced T7 polymerase production levels. Another approach used freshly transformed bacteria to obtain the target protein [5-9]. However, none of these approaches have resolved the problem adequately.…”

Section: Introductionmentioning

confidence: 99%

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A novel system for stable, high-level expression from the T7 promoter

et al. 2012

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BackgroundThe most widespread, efficient prokaryotic protein-producing system is one where the T7 phage polymerase recognizes the T7 phage promoter (T7 p/p system). Unfortunately, in this system, target protein expression gradually declines and is often undetectable following 3 to 5 subcultures. Although a number of studies have attempted to stabilize the expression levels of the T7 p/p system, none has resolved the problem adequately and thus precludes the use of this system for the production of recombinant proteins on a large scale.ResultsWe created an expression cassette enabling stable, high-level expression in the T7p/p system. The cassette was tested with two different vector backbones and two target proteins. In all experiments, the expression system using the new cassette exhibited high and stable protein expression levels when compared to the traditional system.ConclusionsHerein, we describe a universal expression cassette that enables high-level, stable target protein expression in T7 RNA polymerase-based expression systems. We also present the successful use of this cassette as a novel expression platform and demonstrate its ability to overcome the main deficiency of the T7 p/p system. Thus, we provide a method for using the T7 p/p system on an industrial scale.

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Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Cited by 47 publications

References 11 publications

Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

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

A novel system for stable, high-level expression from the T7 promoter

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