Abstract
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 ΦX174-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.
Highlights
The gram-negative bacterium Escherichia coli is the primary microbial cell factory (Andersen and Krummen 2002; Carneiro et al 2012; Choi et al 2006) producing 39% of all recombinant proteins on the market (Demain and Vaishnav 2009)
We aim to present a generic method for the recovery of recombinant protein from inclusion bodies from highdensity fed-batch processes that is based on the controlled expression of bacteriophage φX174-derived lysis protein E
The findings of E-mediated lysis (E-lysis) efficiencies below 90% at the end of fermentation can be explained by the formation of a subpopulation of viable but non-dividing cells due to the stress the cells undergo during recombinant protein production (Glick 1995)
Summary
The gram-negative bacterium Escherichia coli is the primary microbial cell factory (Andersen and Krummen 2002; Carneiro et al 2012; Choi et al 2006) producing 39% of all recombinant proteins on the market (Demain and Vaishnav 2009). Classical cell rupture methods used to release the protein (soluble or in the form of inclusion body aggregates) from the cytoplasm are the following: detergent treatment, cell rupture by lysozyme, sonication, and freeze-thaw (Bird et al 2004), as well as high-pressure homogenization, bead milling, and thermolysis (Ren et al 2007)
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