A rapid and sensitive enzyme immunoassay for Cu/Zn Superoxide dismutase with polyclonal and monoclonal antibodies

Porstmann, T; Wietschke, R.; Schmechta, H; Grunow, Roland; Porstmann, B; Bleiber, R; Pergande, M; Stachat, S.; Baehr, R. von

doi:10.1016/0009-8981(88)90285-9

Cited by 48 publications

(19 citation statements)

References 16 publications

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“…The recombinant protein was quantified via SOD-ELISA [32], and the ratio between soluble protein and inclusion bodies was determined by SDS-PAGE as previously described [33]. …”

Section: Methodsmentioning

confidence: 99%

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

et al. 2013

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BackgroundIn the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in biotechnology has led to the development of many different strains, and selecting an ideal host to produce a specific protein of interest is an important step in developing a production process. E. coli B and K–12 strains are frequently employed in large-scale production processes, and therefore are of particular interest. We previously evaluated the individual cultivation characteristics of E. coli BL21 and the K–12 hosts RV308 and HMS174. To our knowledge, there has not yet been a detailed comparison of the individual performances of these production strains in terms of recombinant protein production and system stability. The present study directly compared the T7-based expression hosts E. coli BL21(DE3), RV308(DE3), and HMS174(DE3), focusing on evaluating the specific attributes of these strains in relation to high-level protein production of the model protein recombinant human superoxide dismutase (SOD). The experimental setup was an exponential carbon-limited fed-batch cultivation with minimal media and single-pulse induction.ResultsThe host strain BL21(DE3) produced the highest amounts of specific protein, followed by HMS174(DE3) and RV308(DE3). The expression system HMS174(DE3) exhibited system stability by retaining the expression vector over the entire process time; however, it entirely stopped growing shortly after induction. In contrast, BL21(DE3) and RV308(DE3) encountered plasmid loss but maintained growth. RV308(DE3) exhibited the lowest ppGpp concentration, which is correlated with the metabolic stress level and lowest degradation of soluble protein fraction compared to both other strains.ConclusionsOverall, this study provides novel data regarding the individual strain properties and production capabilities, which will enable targeted strain selection for producing a specific protein of interest. This information can be used to accelerate future process design and implementation.

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“…The recombinant protein was quantified via SOD-ELISA [32], and the ratio between soluble protein and inclusion bodies was determined by SDS-PAGE as previously described [33]. …”

Section: Methodsmentioning

confidence: 99%

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

et al. 2013

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“…To estimate the specific GFPmut.3.1 content, off-line fluorescence was measured using a microplate reader (Spectramax Gemini XS; Molecular Devices, Mountain View, CA) and calculated using SoftmaxPro. Recombinant GFP and human SOD content were quantified by ELISA according to Reischer et al (2004) and Porstmann et al (1988). For ELISA quantification two replicates of each sample were analyzed two times and the maximal coefficient of variation of 10% was used as acceptance criterion.…”

Section: Off-line Analysismentioning

confidence: 99%

Plasmid‐free T7‐based Escherichia coli expression systems

Striedner

Pfaffenzeller

Luchner

et al. 2010

Biotech & Bioengineering

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In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing.

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“…Capturing antibody mouse anti-SOD mAb IAM-SOD M05, (200 lg/mL 0.1 M NaHCO 3 , pH 9.6±9.8) was applied onto ELISA plates and kept at 4°C overnight. Several dilutions of bacterial samples were added, incubated for 1 h, washed three times with washing buer (phosphate-buered salines, pH 7.2±7.4) and stained with mouse anti-SOD mAb IAM-SOD A11H4 conjugated with alkaline phosphatase (Porstmann et al, 1988).…”

Section: Process Monitoringmentioning

confidence: 99%

Stabilizing plasmid copy number to improve recombinant protein production

Grabherr

Nilsson

Striedner

et al. 2001

Biotech & Bioengineering

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The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.

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A rapid and sensitive enzyme immunoassay for Cu/Zn Superoxide dismutase with polyclonal and monoclonal antibodies

Cited by 48 publications

References 16 publications

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

Plasmid‐free T7‐based Escherichia coli expression systems

Stabilizing plasmid copy number to improve recombinant protein production

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