In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 73, No. 3 ( 2007-02), p. 906-912
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-α2b), 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-α2b expression remained poor even when fused to a signal sequence, and an alternative IFN-α2b 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.
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.01804-06
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2007
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
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