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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost‐effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.  相似文献   

5.
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.  相似文献   

6.
The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-α -PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L−1 to 850 mg L−1 by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L−1, respectively. Received 26 August 1996/ Accepted in revised form 10 December 1996  相似文献   

7.
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  相似文献   

8.
Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds. In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth.  相似文献   

9.
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.  相似文献   

10.
11.
Apolipoprotein A 1 Milano (ApoA‐1M), the protein component of a high‐density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co‐purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA‐1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process. Biotechnol. Bioeng. 2010; 105: 239–249. © 2009 Wiley Periodicals, Inc.  相似文献   

12.
The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.  相似文献   

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