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Biomedical subjects

W E Bentley

Publications and source records attributed to W E Bentley.

6 recordsLinked to original sources
PubMed1995

The effect of cellular energetics on foreign protein production.

Escherichia coli strain F-122 was used to determine if there are additional physiological effects, other than decreasing energetic efficiency accompanied by the excretion of the acetate, on foreign protein production. This organism was the host for expressing HIV582-beta-galactosidase fusion protein under the control of the trp promoter, with ampicillin resistance. By comparing parallel batch cultures with and without acetate addition, it was found that the presence of acetate in the media did not influence beta-galactosidase activity. In these experiments, it appears that the low protein productivity often observed during acetate formation is the result of inefficient cell metabolism, rather than acetate acting as a specific inhibitor of protein production.

Acetates↗
PubMed1995

Optimization for a recombinant E. coli fed-batch fermentation.

The operating strategy that produces the maximum foreign protein expression for a fed-batch process is desired. This is achieved by using a feasible quadratic programming (FSQP) algorithm with a structured model that describes cell growth and product formation for recombinant E. coli. Optimization calculations for a fed-batch culture have not been performed with a model of this complexity up to this point. A constraint on the maximum cell concentration was included. For a fixed value of batch time, the results show that the optimal time profile of feed flow rate can increase the yield of foreign protein by 12-29% over a constant feed rate policy. Also, it was found that the computation time for the FSQP algorithm can be reduced significantly by considering suboptimal profiles of the feed rate, with a minor effect on calculated protein yield.

Algorithms↗
PubMed1994

Continuous insect cell (Sf-9) culture with aeration through sparging.

The continuous growth of Spodoptera frugiperda Sf-9 cells in a 250-ml blown-glass jacketed spinner flask under a direct air sparging environment was investigated. Even at 220 ml working volume (about 90% of total volume), this spinner flask provided good mixing and oxygenation as demonstrated by a higher cell density compared with fermentor cultures. This eliminates a common limitation of the traditional spinner flask, namely much lower cell density at high working volume. Furthermore, this spinner flask has been run with Sf-9 cell culture at five different dilution rates and two different air sparging rates at steady state, demonstrating its utility in research applications where cell size, metabolic activity and environmental conditions can be constantly maintained. In addition to demonstrating the utility of the reactor, three novel points are made in this report. First, cell density in continuous cultures is increased significantly due to a high agitation rate and, especially, air sparging rate, which is seldom used in animal cell or insect cell culture. Second, there is no apparent difference in the specific death rate at two different sparging rates (0.0093 vvm and 0.0125 vvm). Finally, we have maintained Sf-9 cells for more than 4 months in a continuous culture using a serum-free medium without loss of recombinant protein expression in infected cells.

Aerobiosis↗
PubMedDate not supplied

Effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydrolase production.

The recombinant protein yields for batch cultures of the insect cell baculovirus expression system have been significantly enhanced by oxygen, glucose, and glutamine feeding. The improvement in both volumetric and specific yields was based on influencing the metabolism of infected cells. Oxygen was absolutely required for viral replication and high protein expression in infected cells. Increases of 200% in volumetric yield and 100% in specific yield of recombinant epoxide hydrolase were achieved by controlling the dissolved oxygen (DO) level to near 35% saturation. An additional 100% increase was achieved by glucose and glutamine feeding. Results indicated that the intracellular metabolite pool was not adequate for recombinant protein overproduction. Finally, the specific protein yield, based on initial infection cell density, in high cell density spinner flasks and bioreactors of spent media with glucose and glutamine feeding was equivalent to that freshly diluted cultures.

Animals↗