Limits...
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.

Show MeSH

Related in: MedlinePlus

SDS-PAGE analysis of protein expression.(A) Protein expression in shake flask cultures. MW, molecular weight standards; DM, defined medium; YT, complex medium (2xYT) and purified VCL protein standards 0.7 to 2.0 μg. (B) Protein expression time course, fed-batch process with stepwise temperature reduction after induction. MW, molecular weight standards; I, pre-induction; I + 1 to I + 24 hours after induction; VCL, purified VCL standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539881&req=5

Figure 2: SDS-PAGE analysis of protein expression.(A) Protein expression in shake flask cultures. MW, molecular weight standards; DM, defined medium; YT, complex medium (2xYT) and purified VCL protein standards 0.7 to 2.0 μg. (B) Protein expression time course, fed-batch process with stepwise temperature reduction after induction. MW, molecular weight standards; I, pre-induction; I + 1 to I + 24 hours after induction; VCL, purified VCL standard.

Mentions: In the present study, recombinant bacterial collagen, VCL, has been expressed using the pCold vector system, as it had previously been shown that this system was effective in shaker culture for production of the protein in the E .coli host strain BL21 [18,25]. The construct was termed VCL, where V is the non-triple-helical registration domain and CL is the triple-helical, collagen-like domain (Figure 1). The pCold vectors selectively induce target protein synthesis at low temperatures where the synthesis of host proteins is reduced or suppressed and protease activity is decreased. The pCold system is designed for efficient protein expression utilising a promoter derived from the cspA gene, which is a cold-shock gene, with a lac operator inserted downstream to control expression [26]. In addition, expression of a second construct, VCLH, which included a heparin binding domain, H, substituted using site directed mutagenesis into the CL sequence, was also tested. In this construct, a heparin binding sequence, GRPGKRGKQGQK, derived from the collagenous tail of acetylcholine esterase [23,24] was substituted via PCR directed integration into the CL sequence GPAGPMGPAGER that starts at base pair 564/amino acid residue 188 (Figure 1). The substitution was confirmed by DNA sequencing. The pCold vector system is available as a research tool; it is possible that proprietary commercial vectors could give much better yields than observed in the present study that has used the pCold system. Recombinant proteins, VCL and VCLH, were isolated in sufficient quantities for use as electrophoretic quantitation standards (Figure 2A) using a conventional purification protocol (Ni metal affinity column chromatography and with binding via a His6 tag incorporated at the N-terminus of the protein). Final purification was achieved by gel permeation chromatography [25].


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)

SDS-PAGE analysis of protein expression.(A) Protein expression in shake flask cultures. MW, molecular weight standards; DM, defined medium; YT, complex medium (2xYT) and purified VCL protein standards 0.7 to 2.0 μg. (B) Protein expression time course, fed-batch process with stepwise temperature reduction after induction. MW, molecular weight standards; I, pre-induction; I + 1 to I + 24 hours after induction; VCL, purified VCL standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: SDS-PAGE analysis of protein expression.(A) Protein expression in shake flask cultures. MW, molecular weight standards; DM, defined medium; YT, complex medium (2xYT) and purified VCL protein standards 0.7 to 2.0 μg. (B) Protein expression time course, fed-batch process with stepwise temperature reduction after induction. MW, molecular weight standards; I, pre-induction; I + 1 to I + 24 hours after induction; VCL, purified VCL standard.
Mentions: In the present study, recombinant bacterial collagen, VCL, has been expressed using the pCold vector system, as it had previously been shown that this system was effective in shaker culture for production of the protein in the E .coli host strain BL21 [18,25]. The construct was termed VCL, where V is the non-triple-helical registration domain and CL is the triple-helical, collagen-like domain (Figure 1). The pCold vectors selectively induce target protein synthesis at low temperatures where the synthesis of host proteins is reduced or suppressed and protease activity is decreased. The pCold system is designed for efficient protein expression utilising a promoter derived from the cspA gene, which is a cold-shock gene, with a lac operator inserted downstream to control expression [26]. In addition, expression of a second construct, VCLH, which included a heparin binding domain, H, substituted using site directed mutagenesis into the CL sequence, was also tested. In this construct, a heparin binding sequence, GRPGKRGKQGQK, derived from the collagenous tail of acetylcholine esterase [23,24] was substituted via PCR directed integration into the CL sequence GPAGPMGPAGER that starts at base pair 564/amino acid residue 188 (Figure 1). The substitution was confirmed by DNA sequencing. The pCold vector system is available as a research tool; it is possible that proprietary commercial vectors could give much better yields than observed in the present study that has used the pCold system. Recombinant proteins, VCL and VCLH, were isolated in sufficient quantities for use as electrophoretic quantitation standards (Figure 2A) using a conventional purification protocol (Ni metal affinity column chromatography and with binding via a His6 tag incorporated at the N-terminus of the protein). Final purification was achieved by gel permeation chromatography [25].

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