Abstract
BackgroundEscherichia coli (E. coli) is the most abundant expression host for recombinant proteins. The production efficiency is dependent on a multitude of parameters. Therefore, high-throughput applications have become an increasingly frequent technique to investigate the main factors. Within this study, the effects of temperature, induction time and inducer concentration on the metabolic state and the product formation were extensively examined. Induction profiling of E. coli Tuner(DE3) pRhotHi-2-EcFbFP was performed in 48-well Flowerplates and standard 96-well plates using a robotic platform. In parallel shake flask cultivations, the respiration activity of the microorganisms was analyzed. Therefore, two online-monitoring systems were applied: the BioLector for microtiter plates and the RAMOS-device for shake flasks. The impact of different induction conditions on biomass and product formation as well as on the oxygen transfer rate was surveyed.ResultsDifferent optimal induction conditions were obtained for temperatures of 28, 30, 34, and 37 °C. The best inducer concentrations were determined to be between 0.05 and 0.1 mM IPTG for all investigated temperatures. This is 10–20 times lower than conventional guidelines suggest. The induction time was less relevant when the correct inducer concentration was chosen. Furthermore, there was a stronger impact on growth and respiration activity at higher temperatures. This indicated a higher metabolic burden. Therefore, lower IPTG concentrations were advantageous at elevated temperatures. Very similar results were obtained in standard 96-well plates.ConclusionTwo online-monitoring systems were successfully used to investigate the optimal induction conditions for the E. coli Tuner(DE3) pRhotHi-2-EcFbFP strain (lacY deletion mutant) at four different temperatures. The experimental effort was reduced to a minimum by integrating a liquid handling robot. To reach the maximum product formation, a detailed induction analysis was necessary. Whenever the cultivation temperature was changed, the induction conditions have to be adapted. Due to the experimental options provided by the BioLector technology, it was found that the higher the cultivation temperature, the lower the inducer concentration that has to be applied.
Highlights
Escherichia coli (E. coli) is the most abundant expression host for recombinant proteins
In 1982, the recombinant protein technology achieved the great breakthrough with Humulin, a recombinant human insulin expressed in Escherichia coli (E. coli), which was the first approved biopharmaceutical [1]
Comparable oxygen supply in microtiter plates and shake flasks The combination of Respiration Activity Monitoring System (RAMOS) and BioLector onlinemonitoring technologies provide broad information of investigated cultivations
Summary
Escherichia coli (E. coli) is the most abundant expression host for recombinant proteins. Recombinant protein production always implicates a perturbation of the host cell metabolism, which typically leads to lower biomass growth rates [4,5,6]. This stems from high maintenance requirements for the replication. Many parameters have to be individually optimized This involves the choice of vector and promoter system, and composition of the medium, oxygen supply, cultivation temperature and induction conditions [3, 10, 13,14,15,16,17]
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