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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.

Abstract：

: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-alpha2b), 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-alpha2b expression remained poor even when fused to a signal sequence, and an alternative IFN-alpha2b 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.

journal_name

Appl Environ Microbiol

journal_title

Applied and environmental microbiology

authors

Sletta H,Tøndervik A,Hakvåg S,Aune TE,Nedal A,Aune R,Evensen G,Valla S,Ellingsen TE,Brautaset T

doi

10.1128/AEM.01804-06

subject

Has Abstract

pub_date

2007-02-01 00:00:00

pages

906-12

issue

eissn

0099-2240

issn

1098-5336

pii

AEM.01804-06

journal_volume

pub_type

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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.