Results and Applications
EnBase Flo has also been shown to improve protein yields in shake flasks and 24-deep-well plates. In three different protein examples, cell densities corresponding to 10–15 g/L cell dry weight and 10-fold higher volumetric yields of soluble recombinant proteins were compared to standard cultures in LB, TB, and mineral-salt medium.
The product’s well-balanced combination of mineral salts and complex additives provides an optimal composition and stable pH environment for improved protein production.
Cultivation consists of three steps: (1) controlled overnight growth in fed-batch mode to OD600 of 10; (2) addition of an inducer together with complex additives acting as growth booster and pH stabilizer, providing efficient protein synthesis within 3 to 6 hours; and (3) a growth period of 16 to 24 hours characterized by balanced protein synthesis and folding.
Cultivation media for high-throughput applications must meet specific criteria to be selected as the preferred medium of choice—easy and fast preparation, long duration stability, provision of ideal cultivation conditions for high cell density, and high protein productivity per cell.
EnBase is supplied either as ready-to-use liquid or as sterile tablets. EnPresso tablets are designed to be dissolved in 50 mL of sterile water and are useful for shake-flask applications providing protein yields of approximately 10 to 50 mg.
Figure 2 illustrates the EnPresso cultivation process. Two media tablets are added to 50 mL of sterile water in a 500 mL shake flask together with the delivered enzyme, cells, and antibiotics. After at least 12 hours of cultivation, the inducer is added with the enzyme and the booster tablet. The cultivation continues for up to 24 hours, after which the cells are harvested.
Cell growth and recombinant expression of alcohol dehydrogenase from Lactobacillus and multifunctional enzyme type 2 (MFE2) from Drosophila in E. coli in shake flask cultures are shown in Figure 3. Induction of 0.4 mM IPTG is indicated with a dashed vertical line. The graph of EnBase cultivation shows that final OD600 of 32 is obtained with expression of ADH in 50 mL shake-flask cultivations.
Remarkably, only cultures with EnBase were able to ensure the maintenance of favorable pH during the extended induction period (up to 24 hours after induction). Stable pH is one reason for the improved protein production clearly visible in Figure 3.
The impact of various aeration conditions is shown in results from different cultivation formats. This is visible in the ADH-producing strain grown in 24-deep-well plates, which reached a final OD600 of 51 compared to OD600 of 32 in shake flasks. Likewise, the MFE2-producing strain reached OD600 of 31 compared to OD600 of 24 in shake flasks.
Additional testing using Ultra Yield Flasks has shown some promising results with newly designed seals that facilitate optimal gas exchange. Increased mixing and oxygenation resulted in new cultivation records when RB 791 was cultivated with EnPresso: OD600 of 78 and cell dry weight of 20 g/L was achieved. This information verifies that proper aeration is important and the combination of EnBase and Ultra Yield Flasks provides the best results.
Controlled cell growth of bacterial cultures at small scale is beneficial for automated high-throughput methods. The constant glucose feed under growth-limiting conditions enables reliable, reproducible cultivation characteristics. A controlled glucose supply provides a longer exponential cell growth phase, which in practice allows a wide time frame for induction.
Traditionally applied cultivation methods using IPTG induction are fixed to an induction time with low cell densities of about OD600 of 0.6. The alternative induction method using lactose as an inducer, called autoinduction, makes the addition of an inducer unnecessary. Unfortunately, the reported expression yield is not adequate in all cases, as the strong induction might lead to a high level of aggregated protein amounts.
The use of EnBase Flo or EnPresso saves both time and labor due to higher volumetric yields. When lower culture volume is needed, it significantly reduces the amount of time and effort required for downstream processing or process optimization. This system could potentially replace standard shake-flask cultivations in the near future.