Recombinant protein secretion in Escherichia coli

Mergulhão, Filipe; Summers, David; Monteiro, Gabriel A.

doi:10.1016/j.biotechadv.2004.11.003

Cited by 426 publications

(357 citation statements)

References 229 publications

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“…This is a posttranslational pathway where the polypeptide is translocated in an unfolded state that is not dependent on the signal recognition particle (13,17). CSP was designed by us based on its amino acid sequence (26), and it was not optimized regarding formation of mRNA secondary structures.…”

Section: Discussionmentioning

confidence: 99%

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

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

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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-␣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-␣2b expression remained poor even when fused to a signal sequence, and an alternative IFN-␣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.

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Section: Discussionmentioning

confidence: 99%

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

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

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“…In most cases, targeting protein to the periplasmic space or to the culture medium facilitates downstream processing, folding and in vivo stability, enabling the production of soluble and biologically active proteins at a reduced process cost (Mergulhao et al 2005). Excretory overexpression of CGTase from Bacillus sp.…”

Section: Introductionmentioning

confidence: 99%

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

et al. 2011

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Abstract:The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24• C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

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“…Soluble recombinant proteins that are secreted into the medium or into the periplasmic space are even more readily recovered. E. coli has two types of secretion mechanisms that can be used to secrete a number of native proteins (Mergulhão et al, 2005). The type-I mechanisms export high molecularweight toxins and exoenzymes to the culture medium (Fernandez and de Lorenzo, 2001), whereas the type-II mechanisms utilize a two-step process for extracellular secretion in which periplasmic translocation (Koster et al, 2000) is followed by outer membrane translocation.…”

Section: Introductionmentioning

confidence: 99%

“…We surmised that each such curve was derived from a different periplasmic translocation (secretion) pathway involving an inner membrane channel that accommodates a specific range of N-terminal pI values (acidic, neutral, or alkaline) (Lee et al, 2010). However, E. coli type-II cytoplasmic membrane translocation pathways for soluble expression are very complex (Mergulhão et al, 2005), leading to the conclusion that the presently known pathways are insufficient to explain the mechanism of cytoplasmic membrane translocation for soluble expression. We previously proposed that the pI value of the short Nterminal of the signal sequence could represent the whole length as a directional signal (Lee et al, 2008a).…”

Section: Introductionmentioning

confidence: 99%

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

Lee

Han

Kim

et al. 2011

Molecules and Cells

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Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔG RNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.

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Recombinant protein secretion in Escherichia coli

Cited by 426 publications

References 229 publications

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

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

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

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