Volume 5 Supplement 1

The 4th Recombinant Protein Production Meeting: a comparative view on host physiology

Open Access

Pseudomonas fluorescens – a robust expression platform for pharmaceutical protein production

  • Diane Retallack1,
  • J Carrie Schneider1,
  • Lawrence Chew1,
  • Tom Ramseier1,
  • Jeffrey Allen1,
  • Anant Patkar1,
  • Charles Squires1,
  • Henry Talbot1 and
  • Jon Mitchell1
Microbial Cell Factories20065(Suppl 1):S28

https://doi.org/10.1186/1475-2859-5-S1-S28

Published: 10 October 2006

Background

A bottleneck to protein pharmaceutical production can be efficient expression of the target protein. A Pseudomonas fluorescens-based manufacturing platform for high yield production of non-glycosylated protein pharmaceuticals has been developed. This platform is derived from P. fluorescens biovar I strain MB101 [1]. The system's performance is due to the combination of a robust host strain, the availability of extensive molecular biology and bioinformatics tools, and a well optimized high cell density fermentation process. The Systems Biology tools include a genomics and functional genomics capability, a range of stable plasmid vectors of various copy numbers, non-antibiotic-dependent plasmid maintenance [2], multiple expression cassettes [3] and engineered host strains for stringent control of gene expression, and the ability to export proteins to the cell's periplasmic space.

Results

The ability to export proteins to the periplasmic space enables the formation of a precise N-terminus and formation of disulfide bonds. Moreover, export to the periplasm can simplify downstream processing. Multiple native P. fluorescens secretion leader sequences have been evaluated for the ability to direct heterologous proteins to the periplasm. Several secretion leaders were shown to effectively enable secretion of recombinant proteins with precise N-terminal processing at the expected amino acid. Yields of up to 18 g/L of secreted protein have been observed at the 20 L fermentation scale.

Conclusion

P. fluorescens is a robust expression host for high yield expression of secreted proteins. The identification of multiple, effective secretion leaders that can direct secretion of high levels of protein to the periplasm offers flexibility to identify the best secretion leader for each recombinant protein.

Authors’ Affiliations

(1)
Biotechnology R&D, Dowpharma, The Dow Chemical Company

References

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  2. Schneider JC, Jenings AF, Mun DM, McGovern PM, Chew LC: Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation. Biotechnol Prog. 2005, 21 (2): 343-348. 10.1021/bp049696g.View ArticleGoogle Scholar
  3. Retallack DM, Thomas TC, Shao Y, Haney KL, Resnick SM, Lee VD, Squires CH: Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions. Microb Cell Fact. 2006, 5 (1): 1- 10.1186/1475-2859-5-1.View ArticleGoogle Scholar

Copyright

© Retallack et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

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