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

Abstract

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.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.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0832-4) contains supplementary material, which is available to authorized users.

Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden

“…The mastermix was thereupon distributed among the cultivation systems. The comparability of the results from the RAMOS and the BioLector devices at the here used cultivation conditions was already demonstrated elsewhere . Apart from temperature, the shake flask cultivation conditions were identical to those of the second preculture.…”

“…A complete induction profiling was conducted during the main cultivation in one 48‐well MTP using up to 48 different induction conditions. The induction was performed automatically by a liquid handling robot for all Flowerplate cultivations with the so‐called RoboLector platform using a previously presented method . The roundwell plate which was used in the BioLector‐μRAMOS device was induced manually.…”

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

“…In particular, at the time of induction with IPTG after 4 h of cultivation, the measured biomass concentrations are highly diverse. This is relevant since biomass concentration and metabolic status of the culture at the time of induction have a huge influence on the performance of a strain . In comparison, the main cultures produced with a preceding fed‐batch preculture (Figure B) display equalized growth behavior for all 96 cultivations, especially during the first 4 h. As a result, at the time of induction, a comparable concentration of biomass was present in each well, with each strain being in a similar metabolic state.…”

“…However, the metabolic state of cultures at the time of induction is one of the key determining factors for product formation. Thus, diverging cell statuses will lead to unreliable results . Based on such results, it is not possible to evaluate a clone bank with respect to the most productive strain.…”

Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

“…Time of induction and inducer concentration is known to have a high impact on recombinant protein expression . Since secretion of HlyA1 is well controlled by addition of arabinose, time of induction and inducer concentration were varied to study the effect on the respiration behavior of E. coli and the HlyA1 concentration in the supernatant.…”