Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures.

BACKGROUND

Escherichia 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.

RESULTS

Different 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.

CONCLUSION

Two 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.