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Applied and Environmental Microbiology, February 2007, p. 906-912, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01804-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli{triangledown}

H. Sletta,1 A. Tøndervik,2 S. Hakvåg,2 T. E. Vee Aune,2 A. Nedal,2 R. Aune,1 G. Evensen,3 S. Valla,2 T. E. Ellingsen,1 and T. Brautaset1*

SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, Trondheim, Norway,1 Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway,2 Alpharma AS, Oslo, Norway3

Received 31 July 2006/ Accepted 18 November 2006

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-{alpha}2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-{alpha}2b expression remained poor even when fused to a signal sequence, and an alternative IFN-{alpha}2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

* Corresponding author. Mailing address: SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, Sem Selands vei 2, 7465 Trondheim, Norway. Phone: 47 98 28 39 77. Fax: 47 73 59 69 95. E-mail: trygve.brautaset{at}sintef.no.

{triangledown} Published ahead of print on 1 December 2006.

Applied and Environmental Microbiology, February 2007, p. 906-912, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01804-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bakke, I., Berg, L., Aune, T. E. V., Brautaset, T., Sletta, H., Tondervik, A., Valla, S. (2009). Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression. Appl. Environ. Microbiol. 75: 2002-2011 [Abstract] [Full Text]  
  • Xin, W., Wanda, S.-Y., Li, Y., Wang, S., Mo, H., Curtiss, R. III (2008). Analysis of Type II Secretion of Recombinant Pneumococcal PspA and PspC in a Salmonella enterica Serovar Typhimurium Vaccine with Regulated Delayed Antigen Synthesis. Infect. Immun. 76: 3241-3254 [Abstract] [Full Text]  


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