Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation

Saccharomyces cerevisiae was cultivated in a 4-m(3) pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m(3) volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse-shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.     

Characterization of a pilot plant airlift tower loop reactor: III. Evaluation of local properties of the dispersed gas phase during yeast cultivation and in model media

The local properties of the dispersed gas phase (gasholdup, bubble diamater, and bubble velocity) were measured and evaluated at different positions in the riser and downcomer of a pilot plant reactor and, for comparison, in a laboratory reactor. These were described in Parts I and II of this series of articles during yeast cultivation and with model media. In the riser of the pilot plant reactor, the local gas holdup and bubble velocities varied only slightly in axial direction. The gas holdup increased considerably, while the bubble velocity increased only slightly with aeration rate. The bubble size diminished with increasing distance from the aerator in the riser, since the primary bubble size was larger than the equilibrium bubble size. In the downcomer, the mean bubble size was smaller than in the riser. The mean bubble size varied only slightly, the bubble velocity was accelerated, and the gas holdup decreased from top to bottom in the downcomer. In pilot plant at constant aeration rate, the properties of the dispersed phase were nearly constant during the batch cultivation, i.e., they depended only slightly on the cell concentration. In the laboratory reactor, the mean bubble sizes were much larger than in the pilot plant reactor. In the laboratory reactor, the bubble velocities in the riser and downcomer increased, and the mean gas holdup and bubble diameter in the downcomer remained constant as the aeration rate was increased.     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.     

Combined effect of protein fusion and signal sequence greatly enhances the production of recombinant human GM-CSF in Escherichia coli

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor, that has been used as a therapeutic agent in facilitating bone marrow and stem cell transplantation and in other clinical cases like neutropenia. Although biologically active recombinant GM-CSF has been successfully produced in Escherichia coli, the reported levels are extremely poor. In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. This combination had a dramatic effect on expression levels, which increased from 0.8 μg/mL in the control to 40 μg/mL. When a larger fusion partner, such as the Maltose-binding protein (MBP-tag), was used the expression levels increased further to 69.5 μg/mL, which along with the MBP-tag represented approx 12% of the total cellular protein.     

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.