Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins

Alfasi, Sara; Sevastsyanovich, Yanina R.; Zaffaroni, Lenny; Griffiths, Lesley; Hall, Richard M.; Cole, Jeffrey A.

doi:10.1016/j.jbiotec.2011.08.016

Cited by 22 publications

(18 citation statements)

References 49 publications

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“…Surprisingly, there are only two reported examples of evolved protein production strains whose performance was also tested in a culturing setup resembling one often used in industry [134, 146]. If an E. coli protein production strain performs well in small batch culture, it will not necessarily also perform well in more industrial settings, like high cell density fermentations [108].…”

Section: Discussionmentioning

confidence: 99%

“…Note that the term region was chosen to account for all four mutations. In different BL21(DE3)-derived protein production strains including C41(DE3) [101, 142, 143, 146], P lac UV5 has reverted to a weaker variant, designated P lac Weak [143]. This variant still harbours the altered CRP–cAMP binding site of the P lac UV5 region, but reverted to P lac WT in the −10 and the O1-operator sitePicture was taken from [143] with permission …”

Section: E Coli As a Platform For The Production Of Recombinant Protmentioning

confidence: 99%

“…The Cole laboratory isolated BL21(DE3)-derived mutants with an improved ability to produce the cytoplasmic E. coli protein CheY, which is involved in chemotaxis [146]. To facilitate monitoring protein production levels during the isolation process, CheY was fused to GFP.…”

Section: E Coli As a Platform For The Production Of Recombinant Protmentioning

confidence: 99%

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Isolating Escherichia coli strains for recombinant protein production

Schlegel

Genevaux

Gier

2016

Cell. Mol. Life Sci.

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Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.

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Section: Discussionmentioning

confidence: 99%

Section: E Coli As a Platform For The Production Of Recombinant Protmentioning

confidence: 99%

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Isolating Escherichia coli strains for recombinant protein production

Schlegel

Genevaux

Gier

2016

Cell. Mol. Life Sci.

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“…On the transformation plates there was a large number of IPTG resistant BL21(DE3) derivatives. To test if any of these produced YidC-GFP in the cytoplasmic membrane colonies were illuminated with UV light using a standard UV-transilluminator to monitor GFP fluorescence37. In our screen all IPTG-resistant colonies, but one, were not fluorescent upon illumination with UV light.…”

Section: Methodsmentioning

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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“…Among others, fusions with the glia cell stimulating factor (GSF) were used for improving the production of different target recombinant proteins. 76 The aquaporin Z (AqpZ) expression was improved by fusion with TrxA in E. coli. 77 The expression of recombinant proteins as fusions with the small ubiquitin-related modifier (SUMO) protein significantly increased the yield of difficult-to-express proteins in E. coli.…”

Section: Strategies For Achieving High-level Expression Of Microbial mentioning

confidence: 99%

How to achieve high-level expression of microbial enzymes

et al. 2013

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Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field.

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Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins

Cited by 22 publications

References 49 publications

Isolating Escherichia coli strains for recombinant protein production

Isolating Escherichia coli strains for recombinant protein production

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

How to achieve high-level expression of microbial enzymes

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