Dataset Information

Boosting Recombinant Inclusion Body Production-From Classical Fed-Batch Approach to Continuous Cultivation.

ABSTRACT: State of the art microbial recombinant protein production is regularly performed in fed-batch based cultivations. However, these cultivations suffer from highly time-dependent changes in productivity and product quality, leading to high variations in the downstream process. Continuous biomanufacturing offers the possibility of a time independent process, boosting the time-space-yield of the recombinantly produced protein and further reducing costs for production, also as downstream gets more predictive. In the current work, the continuous production of a pharmaceutically relevant protein in form of an inclusion body in E. coli BL21(DE3) was investigated in single vessel cultivations by varying dilution rates using glycerol as carbon source, inducer (lactose or IPTG) and respective inducer concentrations. Attempts to increase low specific productivities observed in single vessel continuous cultivations, led to the establishment of a continuously operated cascade of two stirred tank reactors to spatially separate biomass formation from recombinant protein production. Process performance was substantially improved compared to a single vessel chemostat culture, as specific productivity and space-time yield were boosted using an optimized cascaded process by about a factor of 100. This study shows the potential of a two-stage continuous process as promising alternative to benchmark fed-batch processes achieving constant inclusion body production at a time-independent level.

SUBMITTER: Kopp J

PROVIDER: S-EPMC6834550 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Boosting Recombinant Inclusion Body Production-From Classical Fed-Batch Approach to Continuous Cultivation.

Kopp Julian J Kolkmann Anna-Maria AM Veleenturf Patrick Gwen PG Spadiut Oliver O Herwig Christoph C Slouka Christoph C

Frontiers in bioengineering and biotechnology 20191031

State of the art microbial recombinant protein production is regularly performed in fed-batch based cultivations. However, these cultivations suffer from highly time-dependent changes in productivity and product quality, leading to high variations in the downstream process. Continuous biomanufacturing offers the possibility of a time independent process, boosting the time-space-yield of the recombinantly produced protein and further reducing costs for production, also as downstream gets more pre ...[more]

PMID: 31737617

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Project description:Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ?X174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h-1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of -0.8 ± 0.3 h-1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.

Dataset Information

Boosting Recombinant Inclusion Body Production-From Classical Fed-Batch Approach to Continuous Cultivation.

Publications

Boosting Recombinant Inclusion Body Production-From Classical Fed-Batch Approach to Continuous Cultivation.

Similar Datasets

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