Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Baumann, Pascal; Bluthardt, Nicolai; Renner, Sarah W.; Burghardt, Hannah; Osberghaus, Anna; Hubbuch, Jürgen

doi:10.1016/j.jbiotec.2015.02.024

Cited by 14 publications

(18 citation statements)

References 44 publications

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“…To allow the investigation of different factors, a screening was conducted using the BioLector ® microfermentation system (m2p-labs, Germany). Baumann et al also investigated various cultivation conditions including the oxygen transfer rate by varying the shaking speed as well as the inducer concentration (Baumann et al 2015). Since these experiments showed no influence on bacterial growth and product formation after variation of IPTG concentration, but very high product concentrations were observed without induction of the T7-promoter, in this work only induction with 1 mM IPTG as well as no induction were conducted.…”

Section: Resultsmentioning

confidence: 99%

“…This is consistent with the findings of Baumann et al, who showed that variation of the inductor concentration had no distinctive influence on bacterial growth and protein expression of the investigated target molecule. The same applied to the cultivation temperature, while the shaking speed was found to be more influencing concerning these matters (Baumann et al 2015). On the basis of these investigations, experiments under oxygen deficiency have been carried out.…”

Section: Discussionmentioning

confidence: 99%

“…The determined specific activities of the d -Hyd(co) are in each case much higher than those of the d -Hyd, which confirms this assumption. It was observed that the specific enzyme activity of the d -Hyd(co) increased with decreasing rotation speed, which certainly results from slower growth rates as well as expression which is accompanied by correct folding of the enzyme (Strandberg and Enfors 1991; Baumann et al 2015). Georgiou et al reported that the time-scale for the folding of proteins may vary from milliseconds to days, depending on the amount of kinetic barriers that are included in the folding process (Georgiou and Valax 1996).…”

Section: Discussionmentioning

confidence: 99%

“…This is a fact, although the induction was conducted in a later growth phase (OD 600 = 4) according to Baumann et al, since earlier induction would have led to faster protein expression and therefore also fast and poor folding of the target molecule (Georgiou et al 1994; Georgiou and Valax 1996; Baumann et al 2015). The d -Hyd of non-induced cultures showed a significant decrease in specific activities compared to that of induced cultures, while for the d -Hyd(co) of non-induced cultures only a slight decrease in specific activity was observed.…”

Section: Discussionmentioning

confidence: 99%

“…This suggests that combining both parameters that cause chemical stress affected the proliferation as well as protein expression so much that potentially improved folding of the target molecule carried no weight. When comparing maximum growth rates and specific activities, it was shown that the theory of a more correct protein folding at lower maximum growth rates and therefore lower expression rates (Baumann et al 2015), was only consistent for the codon-optimized hydantoinase d -Hyd(co), which generally exhibits higher expression levels.…”

Section: Discussionmentioning

confidence: 99%

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Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

et al. 2017

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The hydantoinase process is applied for the industrial synthesis of optically pure amino acids via whole cell biocatalysis, providing a simple and well-established method to obtain the catalyst. Nevertheless, whole cell approaches also bear disadvantages like intracellular degradation reactions, transport limitations as well as low substrate solubility. In this work the hydantoinase and carbamoylase from Arthrobacter crystallopoietes DSM 20117 were investigated with respect to their applicability in a cell-free hydantoinase process. Both enzymes were heterologously expressed in Escherichia coli BL21DE3. Cultivation and induction of the hydantoinase under oxygen deficiency resulted in markedly higher specific activities and a further increase in expression was achieved by codon-optimization. Further expression conditions of the hydantoinase were tested using the microbioreactor system BioLector®, which showed a positive effect upon the addition of 3% ethanol to the cultivation medium. Additionally, the hydantoinase and carbamoylase were successfully purified by immobilized metal ion affinity using Ni Sepharose beads as well as by functionalized magnetic beads, while the latter method was clearly more effective with respect to recovery and purification factor. Immobilization of both enzymes via functionalized magnetic beads directly from the crude cell extract was successful and resulted in specific activities that turned out to be much higher than those of the purified free enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0420-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

et al. 2017

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Microscale disruption of microorganisms for parallelized process development

Walther

Dürauer

2017

Biotechnology Journal

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Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris are the standard platforms for biopharmaceutical production with 40% of all between 2010 to 2014 approved protein drugs produced in those microbial hosts. Typically, products overexpressed E. coli and S. cerevisiae remain in the cytosol or are secreted into the periplasm. Consequently, efficient cell disruption is essential for high product recovery during microbial production. Process development platforms at microscale are essential to shorten time to market. While high-pressure homogenization is the industry standard for cell disruption at large scale this method is not practicable for experiments in microscale. This review describes microscale methods for cell disruption at scales as low as 200 µL. Strategies for automation, parallelization and miniaturization, as well as comparability of the results at this scale to high pressure homogenization are considered as those criteria decide which methods are most suited for scale down. Those aspects are discussed in detail for protein overexpression in E. coli and yeast but also the relevance for alternative products and host such as microalgae are taken into account. The authors conclude that bead milling is the best comparable microscale method to large scale high-pressure homogenization and therefore the most suitable technique for automated process development of microbial hosts with the exception of pDNA production.

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Microbioreactor Systems for Accelerated Bioprocess Development

Hemmerich

Noack

Wiechert

et al. 2018

Biotechnology Journal

138

143

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In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

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Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Cited by 14 publications

References 44 publications

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

Microscale disruption of microorganisms for parallelized process development

Microbioreactor Systems for Accelerated Bioprocess Development

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