Parameters Affecting Gene Expression from the Pm Promoter in Gram-Negative Bacteria

Winther‐Larsen, Hanne C.; Josefsen, Kjell D.; Brautaset, Trygve; Valla, Svein

doi:10.1006/mben.1999.0142

Cited by 38 publications

(53 citation statements)

References 62 publications

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“…It has been suggested that this effect is due to lack of proteolysis, but this hypothesis is not yet well confirmed experimentally. We have previously demonstrated that the parental vector pJB658 can be used for high-level expression of different bacterial proteins without the use of any signal sequences (3,4,31). Plasmid pJBphOx-271d and its derivatives constructed in this study have retained the region covering Pm and rbs of pJB658 unmodified, so the observations made here do not seem to be related to the vector system as such.…”

Section: Discussionmentioning

confidence: 70%

“…Two unique restriction sites, SacI and AgeI, were introduced, flanking the xylS coding region of plasmid pJT19bla (31), by site-directed mutagenesis with the mutagenic oligonucleotide pairs sacI-F and sacI-R and ageI-F and ageI-R, respectively. The 1,025-bp SacI-AgeI fragment was subcloned into the corresponding sites of pLITMUS28 (New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

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The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

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Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-␣2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-␣2b expression remained poor even when fused to a signal sequence, and an alternative IFN-␣2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

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Section: Discussionmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

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“…However, we were instead able to use a previously constructed transposon system for complementation. This system is based on the inducible broad-host range Pm promoter previously used to express the luc reporter gene in E. coli (49). Preliminary studies of this system in P. fluorescens showed that expression was very efficient, since it was observed that even in the absence of inducer the luc expression level was similar to that of the corresponding induced E. coli cells (data not shown).…”

Section: Resultsmentioning

confidence: 99%

The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation

et al. 2003

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Bacterial alginates are produced as 1-4-linked ␤-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to ␣-L-guluronic acid. Here we report the isolation of four different epimerizationdefective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

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“…The nahR/Psal and xylS/Pm regulatory systems have been suggested to be alternative expression systems for the expression of heterologous genes on the basis of their tight regulation and the use of inexpensive inducers (18,20,28,30). We previously designed a novel cascade control system that is able to amplify the gene expression capacity (4).…”

mentioning

confidence: 99%

Improvement of Recombinant Protein Yield by a Combination of Transcriptional Amplification and Stabilization of Gene Expression

Cebolla

Royo

Santero

2002

Appl Environ Microbiol

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We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator, xylS2, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the Pm promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.

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Parameters Affecting Gene Expression from the Pm Promoter in Gram-Negative Bacteria

Cited by 38 publications

References 62 publications

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation

Improvement of Recombinant Protein Yield by a Combination of Transcriptional Amplification and Stabilization of Gene Expression

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