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Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

Li Z, Carstensen B, Rinas U - Microb. Cell Fact. (2014)

Bottom Line: Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors.The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors.The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

View Article: PubMed Central - PubMed

Affiliation: Leibniz University of Hannover, Technical Chemistry - Life Science, Hannover, Germany. Zhaopeng.Li@iftc.uni-hannover.de.

ABSTRACT

Background: Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding.

Results: The assembly and application of a simple self-made "smart sustainable bottle" (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors.

Conclusions: The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

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Related in: MedlinePlus

Production of hFGF-2 in a conventional 2-L bioreactor and the SSB system. Autoinduction cultivations were carried out in a 2-L bioreactor (A1-A4) and the SSB system (B1-B4) for the production of hFGF-2. Carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), dissolved oxygen concentration (light gray line), and cell growth (squares) are shown (A1, B1). Moreover, the consumption of glucose (black down triangle), lactose (dark gray up triangle), and glycerol (black circle) as well as the production of acetate (gray diamond) are given (A2, B2). hFGF-2 production in the bioreactor and SSB system was analyzed by SDS-PAGE. W: whole cell protein, S: soluble, and I: insoluble part of whole cell protein (A3, B3). Kinetics of product formation were analyzed by SDS-PAGE of the whole cell protein (A4, B4).
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Fig3: Production of hFGF-2 in a conventional 2-L bioreactor and the SSB system. Autoinduction cultivations were carried out in a 2-L bioreactor (A1-A4) and the SSB system (B1-B4) for the production of hFGF-2. Carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), dissolved oxygen concentration (light gray line), and cell growth (squares) are shown (A1, B1). Moreover, the consumption of glucose (black down triangle), lactose (dark gray up triangle), and glycerol (black circle) as well as the production of acetate (gray diamond) are given (A2, B2). hFGF-2 production in the bioreactor and SSB system was analyzed by SDS-PAGE. W: whole cell protein, S: soluble, and I: insoluble part of whole cell protein (A3, B3). Kinetics of product formation were analyzed by SDS-PAGE of the whole cell protein (A4, B4).

Mentions: Human basic fibroblast growth factor (hFGF-2, 18 kDa) is a cytokine which can be produced as soluble protein but also in form of inclusion bodies depending on the culture conditions employed [5-7]. The comparative production of hFGF-2 was also carried out in the conventional 2-L bioreactor and the SSB system using the autoinduction medium (Figure 3). Again, respiratory activities of the recombinant cells producing hFGF-2 as well as product yields and product solubility were similar in both production vessels (Figure 3, Table 2). Moreover, comparable kinetics of substrates utilization, acetate formation, cell growth and hFGF-2 production were observed in the bioreactor as well as in the SSB system (Figure 3).Figure 3


Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

Li Z, Carstensen B, Rinas U - Microb. Cell Fact. (2014)

Production of hFGF-2 in a conventional 2-L bioreactor and the SSB system. Autoinduction cultivations were carried out in a 2-L bioreactor (A1-A4) and the SSB system (B1-B4) for the production of hFGF-2. Carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), dissolved oxygen concentration (light gray line), and cell growth (squares) are shown (A1, B1). Moreover, the consumption of glucose (black down triangle), lactose (dark gray up triangle), and glycerol (black circle) as well as the production of acetate (gray diamond) are given (A2, B2). hFGF-2 production in the bioreactor and SSB system was analyzed by SDS-PAGE. W: whole cell protein, S: soluble, and I: insoluble part of whole cell protein (A3, B3). Kinetics of product formation were analyzed by SDS-PAGE of the whole cell protein (A4, B4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4226889&req=5

Fig3: Production of hFGF-2 in a conventional 2-L bioreactor and the SSB system. Autoinduction cultivations were carried out in a 2-L bioreactor (A1-A4) and the SSB system (B1-B4) for the production of hFGF-2. Carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), dissolved oxygen concentration (light gray line), and cell growth (squares) are shown (A1, B1). Moreover, the consumption of glucose (black down triangle), lactose (dark gray up triangle), and glycerol (black circle) as well as the production of acetate (gray diamond) are given (A2, B2). hFGF-2 production in the bioreactor and SSB system was analyzed by SDS-PAGE. W: whole cell protein, S: soluble, and I: insoluble part of whole cell protein (A3, B3). Kinetics of product formation were analyzed by SDS-PAGE of the whole cell protein (A4, B4).
Mentions: Human basic fibroblast growth factor (hFGF-2, 18 kDa) is a cytokine which can be produced as soluble protein but also in form of inclusion bodies depending on the culture conditions employed [5-7]. The comparative production of hFGF-2 was also carried out in the conventional 2-L bioreactor and the SSB system using the autoinduction medium (Figure 3). Again, respiratory activities of the recombinant cells producing hFGF-2 as well as product yields and product solubility were similar in both production vessels (Figure 3, Table 2). Moreover, comparable kinetics of substrates utilization, acetate formation, cell growth and hFGF-2 production were observed in the bioreactor as well as in the SSB system (Figure 3).Figure 3

Bottom Line: Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors.The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors.The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

View Article: PubMed Central - PubMed

Affiliation: Leibniz University of Hannover, Technical Chemistry - Life Science, Hannover, Germany. Zhaopeng.Li@iftc.uni-hannover.de.

ABSTRACT

Background: Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding.

Results: The assembly and application of a simple self-made "smart sustainable bottle" (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors.

Conclusions: The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

Show MeSH
Related in: MedlinePlus