Improved Broad-Host-Range RK2 Vectors Useful for High and Low Regulated Gene Expression Levels in Gram-Negative Bacteria

Blatny, Janet Martha; Brautaset, Trygve; Winther‐Larsen, Hanne C.; Karunakaran, Ponniah; Valla, Svein

doi:10.1006/plas.1997.1294

Cited by 161 publications

(115 citation statements)

References 60 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: 71%

“…Despite the many advantages of this organism, high-level heterologous expression is not routinely achieved, typically due to biased codon usage, gene product toxicity, low gene product solubility, mRNA secondary structure formation, and low mRNA stability (15,26). The broadhost-range plasmid pJB658 harbors the inducible Pm/xylS promoter/regulator elements for recombinant expression of cloned genes in a wide range of gram-negative bacteria (3,4). We recently modified this plasmid to express high volumetric yields of secreted recombinant single-chain antibody variable fragment (scFv-phOx) during HCDC of E. coli (26).…”

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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: 71%

mentioning

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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“…DNA fragments for generation of deletions or complementation of mutants were amplified by PCR using Phusion polymerase (Clontech) and primers listed in Table 2. The broad host-range plasmid pJB3Cm6 [30] was used for the complementation experiments. Recombinant plasmids were transferred from E. coli b2155 [31] to S. oneidensis strains by conjugation.…”

Section: Mutagenesis Complementation and Protein Analysis Of S Oneimentioning

confidence: 99%

The Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

et al. 2010

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The ability of the metal reducer Shewanella oneidensis MR-1 to generate electricity in microbial fuel cells (MFCs) depends on the activity of a predicted type IV prepilin peptidase; PilD. Analysis of an S. oneidensis MR-1 pilD mutant indicated that it was deficient in pili production (Msh and type IV) and type II secretion (T2S). The requirement for T2S in metal reduction has been previously identified, but the role of pili remains largely unexplored. To define the role of type IV or Msh pili in electron transfer, mutants that lack one or both pilus biogenesis systems were generated and analyzed; a mutant that lacked flagella was also constructed and tested. All mutants were able to reduce insoluble Fe(III) and to generate current in MFCs, in contrast to the T2S mutant that is deficient in both processes. Our results show that loss of metal reduction in a PilD mutant is due to a T2S deficiency, and therefore the absence of c cytochromes from the outer surface of MR-1 cells, and not the loss of pili or flagella. Furthermore, MR-1 mutants deficient in type IV pili or flagella generated more current than the wild type, even though extracellular riboflavin levels were similar in all strains. This enhanced current generating ability is in contrast to a mutant that lacks the outer membrane c cytochromes, MtrC and OmcA. This mutant generated significantly less current than the wild type in an MFC and was unable to reduce Fe(III). These results indicated that although nanofilaments and soluble mediators may play a role in electron transfer, surface exposure of outer membrane c cytochromes was the determining factor in extracellular electron transfer in S. oneidensis MR-1.

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“…Thus, a synergistic effect could be obtained when activation of the P m by XylS overproduction is further augmented in the presence of inducers like m-toluate. 20 Induction with m-toluate has been used in biotechnological applications such as the production of different enzymes (e.g., the CelB phosphoglucomutase from Acetobacter xylinum 21 or XanA from Xanthomonas campestris, an enzyme involved in xantano biosynthesis).…”

Section: Rationale Of the Transcriptional Cascade Based On Nahr And Xmentioning

confidence: 99%

Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/Psal

2010

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Improved Broad-Host-Range RK2 Vectors Useful for High and Low Regulated Gene Expression Levels in Gram-Negative Bacteria

Cited by 161 publications

References 60 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 Role of Shewanella oneidensis MR‐1 Outer Surface Structures in Extracellular Electron Transfer

Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/Psal

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