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June 01, 2009 (Vol. 29, No. 11)

Controlling Nutrient Release in Cell Cultivation

EnBase Designed to Optimize Feeding in the Production System

  • Recombinant Protein Production

    Click Image To Enlarge +
    Figure 3. Growth curves and target proteins

    Higher volumetric product yields enable scaling down of the protein production process, saving time, space, and manpower. As part of the Human Protein Atlas project, nearly 300 different recombinant proteins (with a range of 25–150 amino acids in length) are produced per week. The protein fragments are fused with an N-terminal hexa-histidine albumin binding protein tag and expressed in E. coli Rosetta (DE3) cells. The cells are cultivated in 100 mL complex medium in 1 L shake flasks.

    To reduce the manual handling steps and increase the throughput, 15 randomly selected clones were tested in EnBase 24 DWP (deep-well plates) using two different EnBase media. With the EnBase DWPs the final cell densities reached an OD600 of 25 (Figure 3A) as compared to an OD600 of 6, which was the average cell density in the shake flasks.

    The protein yields from preliminary data (Figure 3B) obtained with the blue medium looks promising and indicate that one 24 DWP could substitute for six shake flasks.

    Slow controlled cell growth in balanced medium supports the correct folding of translated proteins as shown in Figure 4B.

    Recombinant expression of Pseudozyma antarctica lipase B was investigated by comparison of different expression systems in Pichia pastoris and E. coli strains. Expression (Figure 4) was performed in the periplasmic space of E. coli Rosetta (DE3) using a 1 L shake flask with superbroth medium, which resulted in 4 mg/L of active enzyme.

    The highest enzyme activity value was obtained when EnBase mineral salt medium (MSM) was used for cultivation. High cell density did not guarantee high protein yield, however. Instead the EnBase MSM with 48 U/L amylase showed a yield of 7.0 mg/L (Figure 4A), but had a low cell density of OD600 of 10 (Figure 4B). The higher yield of active enzyme was reached due to a higher amount of soluble protein per cell.

    Efficient small-scale production of recombinant proteins without the use of fermentors affords wide application in structure-based drug design, antibody production, and screening of protein libraries. Screening of different parameters such as strains, vectors with specific promoters, ribosome binding sites, fusion tags, purification tags, media, and additives or induction mode can also be easily performed in parallel in deep-well plates.

    Investigations enabled by Enbase include many process development steps, including determination of glucose gradient by setting glucose pulses, influence of oxygen parameters, or effect of induction time under fed-batch conditions. Although the technology was developed for E. coli, but preliminary studies have shown its feasibility for Bacillus subtilis, Lactobacillus brevis, and Pichia pastoris.

    As the EnBase technology is an appropriate tool for high cell density cultivation in 96 well plates, its application in high-through put screening of metagenomic or mutant libraries is logical. As such, BioSilta has developed a specific EnBase medium for the auxotrophic E. coli mutants DH5 alpha and DH10 beta.

    High-throughput sequencing, high-throughput crystallization approaches, and production of labeled proteins for NMR studies are additional applications.

    One interesting future project is the application of EnBase technology to Wave single-use bioreactors. Cell bags are placed on a special rocking platform to provide mixing and oxygen transfer. As oxygen transfer is limited in Wave bioreactors the EnBase system could control the cell growth and adapt it to the existing oxygen conditions.

  • Click Image To Enlarge +
    Figure 4. Protein yield and cell density of standard and EnBase medium

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