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A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage X174-derived lysis protein E
AuthorEhgartner, Daniela ; Sagmeister, Patrick ; Langemann, Timo ; Meitz, Andrea ; Herwig, Christoph ; Lubitz, Werner
Published in
Applied Microbiology and Biotechnology, 2017, Vol. 101, Issue 14, page 5603-5614
Document typeJournal Article
Keywords (EN)Bioprocess technology / Recombinant protein release / Escherichia coli / pBAD expression system / Mixed feed bioprocesses / Bacterial Ghost
Project-/ReportnumberMorphoplant GmbH, Bochum (RCPE Projekt, 2.29)
URNurn:nbn:at:at-ubtuw:3-4179 Persistent Identifier (URN)
 The work is publicly available
A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage X174-derived lysis protein E [1.52 mb]Supplementary material [0.12 mb]
Abstract (English)

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 h1 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 h1. 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.

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