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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: Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories.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 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
Production of GST-GFP and TRX-hLIF in a conventional 2-L bioreactor and the SSB system with “booster” amino acids addition. Autoinduction cultivations for the production of GST-GFP (A1-A5) and TRX-hLIF (B1-B5) were carried out in 2-L bioreactor (A1 and B1), the SSB system (A2 and B2), and the SSB system with “booster” amino acids addition (A3/4 and B3/4). The carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), and the dissolved oxygen concentration (light gray line) are shown. “Booster” amino acids additions are indicated by arrows in A4 and B4. GST-GFP (A5) and TRX-hLIF (B5) production in a 2-L bioreactor (lanes 1-3), the SSB system (lanes 4-6), and the SSB system with “booster” amino acids addition (lanes 7-9) were analyzed by SDS-PAGE. W: whole cell protein, S: soluble part, and I: insoluble part of whole cell protein.
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Fig4: Production of GST-GFP and TRX-hLIF in a conventional 2-L bioreactor and the SSB system with “booster” amino acids addition. Autoinduction cultivations for the production of GST-GFP (A1-A5) and TRX-hLIF (B1-B5) were carried out in 2-L bioreactor (A1 and B1), the SSB system (A2 and B2), and the SSB system with “booster” amino acids addition (A3/4 and B3/4). The carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), and the dissolved oxygen concentration (light gray line) are shown. “Booster” amino acids additions are indicated by arrows in A4 and B4. GST-GFP (A5) and TRX-hLIF (B5) production in a 2-L bioreactor (lanes 1-3), the SSB system (lanes 4-6), and the SSB system with “booster” amino acids addition (lanes 7-9) were analyzed by SDS-PAGE. W: whole cell protein, S: soluble part, and I: insoluble part of whole cell protein.

Mentions: Some proteins are difficult to produce as soluble bioactive proteins using E. coli as expression system. Examples are the reporter protein GFP carrying an N-terminal glutathione-S-transferase (GST) tag which strongly increases the GFP propensity to form inclusion bodies [3] and the poorly soluble human leukemia inhibitory factor (hLIF) carrying an N-terminal thioredoxin (TRX) tag [8]. The production of both proteins was carried out in the conventional 2-L bioreactor and in the SSB system (Figure 4). Again, the respiratory activities of producing cells were similar in both vessels, however, the production of each protein led to protein-specific respiratory profiles independent of the production vessel (Figure 4). SDS-PAGE analysis revealed very low amounts of both proteins in the soluble cell fraction independent of the production vessel employed (Figure 4).Figure 4


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

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

Production of GST-GFP and TRX-hLIF in a conventional 2-L bioreactor and the SSB system with “booster” amino acids addition. Autoinduction cultivations for the production of GST-GFP (A1-A5) and TRX-hLIF (B1-B5) were carried out in 2-L bioreactor (A1 and B1), the SSB system (A2 and B2), and the SSB system with “booster” amino acids addition (A3/4 and B3/4). The carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), and the dissolved oxygen concentration (light gray line) are shown. “Booster” amino acids additions are indicated by arrows in A4 and B4. GST-GFP (A5) and TRX-hLIF (B5) production in a 2-L bioreactor (lanes 1-3), the SSB system (lanes 4-6), and the SSB system with “booster” amino acids addition (lanes 7-9) were analyzed by SDS-PAGE. W: whole cell protein, S: soluble part, and I: insoluble part of whole cell protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Production of GST-GFP and TRX-hLIF in a conventional 2-L bioreactor and the SSB system with “booster” amino acids addition. Autoinduction cultivations for the production of GST-GFP (A1-A5) and TRX-hLIF (B1-B5) were carried out in 2-L bioreactor (A1 and B1), the SSB system (A2 and B2), and the SSB system with “booster” amino acids addition (A3/4 and B3/4). The carbon dioxide (CTR, black line) and oxygen transfer rates (OTR, gray line), and the dissolved oxygen concentration (light gray line) are shown. “Booster” amino acids additions are indicated by arrows in A4 and B4. GST-GFP (A5) and TRX-hLIF (B5) production in a 2-L bioreactor (lanes 1-3), the SSB system (lanes 4-6), and the SSB system with “booster” amino acids addition (lanes 7-9) were analyzed by SDS-PAGE. W: whole cell protein, S: soluble part, and I: insoluble part of whole cell protein.
Mentions: Some proteins are difficult to produce as soluble bioactive proteins using E. coli as expression system. Examples are the reporter protein GFP carrying an N-terminal glutathione-S-transferase (GST) tag which strongly increases the GFP propensity to form inclusion bodies [3] and the poorly soluble human leukemia inhibitory factor (hLIF) carrying an N-terminal thioredoxin (TRX) tag [8]. The production of both proteins was carried out in the conventional 2-L bioreactor and in the SSB system (Figure 4). Again, the respiratory activities of producing cells were similar in both vessels, however, the production of each protein led to protein-specific respiratory profiles independent of the production vessel (Figure 4). SDS-PAGE analysis revealed very low amounts of both proteins in the soluble cell fraction independent of the production vessel employed (Figure 4).Figure 4

Bottom Line: Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories.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 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