title: 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.
authors.affiliation: SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, Sem Selands vei 2, 7465 Trondheim, Norway.
authors.forename: H
authors.initials: H
authors.lastname: Sletta
authors.forename: A
authors.initials: A
authors.lastname: Tøndervik
authors.forename: S
authors.initials: S
authors.lastname: Hakvåg
authors.forename: T E Vee
authors.initials: TE
authors.lastname: Aune
authors.forename: A
authors.initials: A
authors.lastname: Nedal
authors.forename: R
authors.initials: R
authors.lastname: Aune
authors.forename: G
authors.initials: G
authors.lastname: Evensen
authors.forename: S
authors.initials: S
authors.lastname: Valla
authors.forename: T E
authors.initials: TE
authors.lastname: Ellingsen
authors.forename: T
authors.initials: T
authors.lastname: Brautaset
mesh_terms: D001425:Bacterial Outer Membrane Proteins; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010080:Oxazoles / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins; D011995:Recombination, Genetic
chemical_list: C119678:4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; D001425:Bacterial Outer Membrane Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D010080:Oxazoles; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C078167:OMPA outer membrane proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011133:Polysaccharide-Lyases; C023911:pectate lyase