Effects of Plasmid Copy Number and Runaway Plasmid Replication on Overproduction and Excretion of β‐Lactamase from <i>Escherichia</i> coli

Togna, A. Paul; Shuler, Michael L.; Wilson, David B.

doi:10.1021/bp00019a005

Cited by 38 publications

(32 citation statements)

References 49 publications

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“…Nevertheless, the growth rate of the recombinant strain was lower and statistically diVerent (P < 0.05) for all glucose concentrations compared with that observed for the plasmid-free strain. This growth rate reduction could be explained by plasmid replication and synthesis of plasmid gene products, which require additional energy and material from the host cell [13,47].…”

Section: Resultsmentioning

confidence: 99%

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Rodríguez

Espejo

Hernández

et al. 2010

J Ind Microbiol Biotechnol

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Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

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Section: Resultsmentioning

confidence: 99%

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Rodríguez

Espejo

Hernández

et al. 2010

J Ind Microbiol Biotechnol

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“…In general, presence of non-expressing plasmids results in a decrease in the specific growth rate, the magnitude of this effect depending on the plasmid size and copy number (Birnbaum and Bailey, 1991;Seo and Bailey, 1985). Increases in plasmid copy number as a result of runaway replication can lead to segregational instability (Togna et al, 1993) or even cell death (Nordström et al, 1979). However, in another report presence of a low copy number plasmid encoding multiple antibiotic resistance did not affect the growth rate (Klemperer et al, 1979).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

Rozkov¹,

Avignone-Rossa²,

Ertl³

et al. 2004

Biotech & Bioengineering

106

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The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.

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“…Reports of recombinant proteins in E. coli often describe high levels of export to the periplasm (Champion et al, 2003;Rinas et al, 1993), but there are only a few reports of successful attempts to secrete proteins past the outer membrane (Blight and Holland, 1994;Fu et al, 1993;Togna et al, 1993). Efforts to alleviate bottlenecks in secretion pathways via metabolic engineering do not often produce the desired phenotype, primarily due to the low efficiency and high specificity of most secretion systems and an incomplete understanding of their mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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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Effects of Plasmid Copy Number and Runaway Plasmid Replication on Overproduction and Excretion of β‐Lactamase from Escherichia coli

Cited by 38 publications

References 49 publications

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

Engineering HlyA hypersecretion inEscherichia coli based on proteomic and microarray analyses

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