A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

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.

Keywords: Bacterial Ghost; Bioprocess technology; Escherichia coli; Mixed feed bioprocesses; Recombinant protein release; pBAD expression system.

Fig. 1

An overview a over the feed flow and b biomass concentrations during uninduced and induced fed-batch for three cultivations with different total q _s. The start of induction phase is indicated as time point 0. The gap between uninduced fed-batch and induction phase represents the L-arabinose pulse. In processes with higher q _s, the induction phase was shorter as a biomass concentration of 35 g/l was reached earlier. During uninduced fed-batch, feeding rates were the same for all cultivations, which is shown by the black line

Fig. 2

Online measurements of one cultivation (FB5). a Batch and fed-batch phase. Permittivity signal and CO₂ in the off-gas are shown. Additionally, the biomass estimated by the soft-sensor in the fed-batch phase can be seen. b Induction phase. Estimated biomass is compared to offline biomass measurements. Furthermore, the Permittivity signal and the volumetric product titer are shown. c E-lysis phase. The E-lysis phase begins with the temperature shift. E-lysis efficiency and the Permittivity measurements are shown as well

Fig. 3

Prediction plot for E-lysis efficiency as a function of L-arabinose in the feed and specific growth rate. The red circles indicate the experimental data

Fig. 4

Trend of E-lysis competent cell population during E-lysis phase of FB3. The circles indicate the experimental data while the black line shows the trend

Fig. 5

Prediction of product titer based on the linear regression model for different levels of growth rate and L-arabinose in the feed. The white line indicates the boundary below which 90% of E-lysis efficiency and more could be achieved. The red circles indicate the experimental data

Fig. 6

SDS gel for FB3 and FB5. Lane 1 and 6—standard; FB3: lane 2—last sample before lysis (homogenized pellet), lane 3—sample with highest E-lysis efficiency (pellet): lane 4—homogenization supernatant of the last sample before lysis, lane 5—fermentation supernatant of the last sample before lysis. For FB5, samples were loaded in the same order on lanes 7 to 10. rhBMP-2 was expected at 13 kDa

Fig. 7

Microscope images of empty cell envelopes carrying the inclusion body. a Overview picture. b Picture with higher resolution