Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB

Mergulhão, Filipe; Monteiro, Gabriel A.; Larsson, Gen; Sandén, Anna Maria; Farewell, Anne; Nyström, Thomas; Cabral, J. M. S.; Taipa, M. A.

doi:10.1007/s00253-003-1232-8

Cited by 16 publications

(17 citation statements)

References 24 publications

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“…Escherichia coli transformed with plasmids with different copy numbers exhibited altered export of human proinsulin to the periplasm (Mergulhao et al, 2003). It was found that the use of plasmids of moderate copy number (15 -60) vs. low (10) copy number increased export, but this study did not include a high copy number plasmid.…”

Section: Discussionmentioning

confidence: 97%

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

Lee

2004

Biotechnol. Bioeng.

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Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community.

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Section: Discussionmentioning

confidence: 97%

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

Lee

2004

Biotechnol. Bioeng.

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“…coli to overproduce these bacteriocins. This can be explained by many factors, including the structural features of the recombinant gene sequences, the stability and translational efficiency of the mRNA, incomplete processing of signal sequences, the metabolic burden on the host strain, the capacity of the transport system and the potential toxicity of the bacteriocin 14, 36–38 . Thus, a low copy vector was the most suitable condition for evaluating the expression of both bacteriocins.…”

Section: Discussionmentioning

confidence: 99%

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

Mesa-Pereira

O’Connor

Rea

et al. 2017

Sci Rep

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The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers.

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“…E. coli cells were grown at 37°C in Luria-Bertani (LB) medium (0.5% yeast extract, 1% tryptone, and 0.5% NaCl, pH 7.0). Since complex media such as LB can produce negative effects on the expression of recombinant proteins (Mergulhao et al, 2003), the recombinant E. coli were cultured in M9 medium supplemented with 0.1% glucose, 0.05% yeast extract, 0.05% tryptone, and 100 µg/ml ampicillin at 22°C or 37°C. Induction of each recombinant protein was facilitated by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the optical density at 600 nm (A600) reached 0.4.…”

Section: Al 2006)mentioning

confidence: 99%

Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli

et al. 2008

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An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22 degrees C but was efficiently secreted into the extracellular medium when cultivated at 37 degrees C.

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Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB

Cited by 16 publications

References 24 publications

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli

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