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Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications.

Peng YY, Howell L, Stoichevska V, Werkmeister JA, Dumsday GJ, Ramshaw JA - Microb. Cell Fact. (2012)

Bottom Line: In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins.The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time.These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3168, Australia.

ABSTRACT

Background: Collagen has proved valuable as biomedical materials for a range of clinical applications, particularly in wound healing. It is normally produced from animal sources, such as from bovines, but concerns have emerged over transmission of diseases. Recombinant collagens would be preferable, but are difficult to produce. Recently, studies have shown that 'collagens' from bacteria, including Streptococcus pyogenes, can be produced in the laboratory as recombinant products, and that these are biocompatible. In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins.

Results: Production trials in shake flask cultures gave low yields of recombinant product, < 1 g/L. Increased yields, of around 1 g/L, were obtained when the shake flask process was transferred to a stirred tank bioreactor, and the yield was further enhanced to around 10 g/L by implementation of a high cell density fed-batch process and the use of suitably formulated fully defined media. Similar yields were obtained with 2 different constructs, one containing an introduced heparin binding domain. The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time.

Conclusions: These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.

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Effect of fermentation process design and induction temperature on growth of collagen-producing E. coli strains. Cell concentration was estimated by measuring optical density at 600 nm. See Table 1 for experimental details. (A) Shake flask and low cell density processes.♦, VCL, shake flask, complex medium (2xYT); ▲, VCL, shake flask, defined medium; ■, VCL, bioreactor batch process (low cell density); ●, VCLH, bioreactor batch process (low cell density). (B) High cell density processes. ♦, VCLH, bioreactor fed-batch process, induction at 25°C for 10 hours; ▲, VCL, bioreactor fed-batch process, induction at 25°C for 10 hours; ●, VCL, bioreactor fed-batch process, induction at 15°C for 24 hours; ■, VCL, bioreactor fed-batch process, induction for 24 hours with stepwise temperature reduction.
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Figure 3: Effect of fermentation process design and induction temperature on growth of collagen-producing E. coli strains. Cell concentration was estimated by measuring optical density at 600 nm. See Table 1 for experimental details. (A) Shake flask and low cell density processes.♦, VCL, shake flask, complex medium (2xYT); ▲, VCL, shake flask, defined medium; ■, VCL, bioreactor batch process (low cell density); ●, VCLH, bioreactor batch process (low cell density). (B) High cell density processes. ♦, VCLH, bioreactor fed-batch process, induction at 25°C for 10 hours; ▲, VCL, bioreactor fed-batch process, induction at 25°C for 10 hours; ●, VCL, bioreactor fed-batch process, induction at 15°C for 24 hours; ■, VCL, bioreactor fed-batch process, induction for 24 hours with stepwise temperature reduction.

Mentions: The expression of VCL in shake flasks using 2xYT Media and Defined Media (DM) were used as the baseline process for this study. Cells were grown for 24 h at 37°C, after which an OD600 nm of around 6 was achieved, prior to induction at 25°C. Expressed protein production was determined after incubation at 25°C for 10 h, followed by a further 14 h at 15°C. It was hoped that the additional reduction in temperature would maintain the selective target protein production achieved by the pCold vector system. The changes in cell densities were tracked throughout the process (Figure 3A), and at the end of the production phase, moderate amounts of wet cell paste, 8.3 g/L for DM and 9.7 g/L for 2xYT, were obtained. Expression yields were determined by comparison to known amounts of VCL protein by SDS-PAGE (Figure 2A). These shake flask processes achieved recombinant collagen expression levels of around 0.2 g/L in DM and 0.3 g/L in 2xYT (Table 1). These yields are adequate for biochemical and biophysical studies of VCL and related constructs such as VCLH, but are well below production levels required for commercial production.


Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications.

Peng YY, Howell L, Stoichevska V, Werkmeister JA, Dumsday GJ, Ramshaw JA - Microb. Cell Fact. (2012)

Effect of fermentation process design and induction temperature on growth of collagen-producing E. coli strains. Cell concentration was estimated by measuring optical density at 600 nm. See Table 1 for experimental details. (A) Shake flask and low cell density processes.♦, VCL, shake flask, complex medium (2xYT); ▲, VCL, shake flask, defined medium; ■, VCL, bioreactor batch process (low cell density); ●, VCLH, bioreactor batch process (low cell density). (B) High cell density processes. ♦, VCLH, bioreactor fed-batch process, induction at 25°C for 10 hours; ▲, VCL, bioreactor fed-batch process, induction at 25°C for 10 hours; ●, VCL, bioreactor fed-batch process, induction at 15°C for 24 hours; ■, VCL, bioreactor fed-batch process, induction for 24 hours with stepwise temperature reduction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539881&req=5

Figure 3: Effect of fermentation process design and induction temperature on growth of collagen-producing E. coli strains. Cell concentration was estimated by measuring optical density at 600 nm. See Table 1 for experimental details. (A) Shake flask and low cell density processes.♦, VCL, shake flask, complex medium (2xYT); ▲, VCL, shake flask, defined medium; ■, VCL, bioreactor batch process (low cell density); ●, VCLH, bioreactor batch process (low cell density). (B) High cell density processes. ♦, VCLH, bioreactor fed-batch process, induction at 25°C for 10 hours; ▲, VCL, bioreactor fed-batch process, induction at 25°C for 10 hours; ●, VCL, bioreactor fed-batch process, induction at 15°C for 24 hours; ■, VCL, bioreactor fed-batch process, induction for 24 hours with stepwise temperature reduction.
Mentions: The expression of VCL in shake flasks using 2xYT Media and Defined Media (DM) were used as the baseline process for this study. Cells were grown for 24 h at 37°C, after which an OD600 nm of around 6 was achieved, prior to induction at 25°C. Expressed protein production was determined after incubation at 25°C for 10 h, followed by a further 14 h at 15°C. It was hoped that the additional reduction in temperature would maintain the selective target protein production achieved by the pCold vector system. The changes in cell densities were tracked throughout the process (Figure 3A), and at the end of the production phase, moderate amounts of wet cell paste, 8.3 g/L for DM and 9.7 g/L for 2xYT, were obtained. Expression yields were determined by comparison to known amounts of VCL protein by SDS-PAGE (Figure 2A). These shake flask processes achieved recombinant collagen expression levels of around 0.2 g/L in DM and 0.3 g/L in 2xYT (Table 1). These yields are adequate for biochemical and biophysical studies of VCL and related constructs such as VCLH, but are well below production levels required for commercial production.

Bottom Line: In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins.The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time.These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3168, Australia.

ABSTRACT

Background: Collagen has proved valuable as biomedical materials for a range of clinical applications, particularly in wound healing. It is normally produced from animal sources, such as from bovines, but concerns have emerged over transmission of diseases. Recombinant collagens would be preferable, but are difficult to produce. Recently, studies have shown that 'collagens' from bacteria, including Streptococcus pyogenes, can be produced in the laboratory as recombinant products, and that these are biocompatible. In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins.

Results: Production trials in shake flask cultures gave low yields of recombinant product, < 1 g/L. Increased yields, of around 1 g/L, were obtained when the shake flask process was transferred to a stirred tank bioreactor, and the yield was further enhanced to around 10 g/L by implementation of a high cell density fed-batch process and the use of suitably formulated fully defined media. Similar yields were obtained with 2 different constructs, one containing an introduced heparin binding domain. The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time.

Conclusions: These data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.

Show MeSH
Related in: MedlinePlus