Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach

Aucoin, Marc G.; McMurray-Beaulieu, Virginie; Poulin, Frédéric; Boivin, Eric B; Chen, Jingkui; Ardelean, Francisc M; Cloutier, Michel; Choi, Young Jun; Míguez, Carlos B.; Jolicœur, Mario

doi:10.1186/1475-2859-5-27

Cited by 33 publications

(22 citation statements)

References 20 publications

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“…15 Complex medium on the other hand allows a much higher specific growth rate which in turn enhances specific productivities especially for growth associated product formation. 16 Additionally the presence of amino acids in the medium leads to improved protein yields since amino acid biosynthesis is often a rate limiting step in protein synthesis. 17 Thus most industrial scale bioprocesses prefer the use of complex media to obtain high product titers.…”

Section: Introductionmentioning

confidence: 99%

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

2012

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Fed batch cultures expressing recombinant interferon beta under the T7 promoter were run with different exponential feeding rates of a complex substrate and induced at varying cell densities. Post-induction profiles of the specific product formation rates showed a strong dependence on the specific growth rate with the maximum product yield obtained at 0.2 h(-1). A study of the relative transcriptomic profiles as a function of pre-induction μ was therefore done to provide insight into the role of cellular physiology in enhancing recombinant protein expression. Hierarchical clustering analysis of the significantly regulated genes allowed us to identify biologically important groups of genes which fall under specific master regulators. The groups were: rpoH, ArcB, CreB, Lrp, RelA, Fis and Hfq. The response of these regulators, which exert a feedback control on the growth and product formation rates correlated well with the expression levels obtained. Thus at the optimum pre-induction μ, the alternative sigma factors and ribosomal machinery genes did not get depressed till the 6th hour post-induction unlike at other specific growth rates, demonstrating a critical role for the genes in sustaining recombinant protein expression.

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Section: Introductionmentioning

confidence: 99%

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

2012

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“…Imaging protein expression is important for optimizing production of recombinant proteins 13 . Optimizing protein yield involves screening mutant or genetically modified bacteria for increased protein expression 14-16 .…”

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confidence: 99%

Imaging bacterial protein expression using genetically encoded RNA sensors

2013

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We show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins, and exhibit fluorescence increases that enable protein expression to be imaged in living cells. These sensors provide a general strategy to image protein expression in single bacteria in real-time.

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“…In agreement with this, the use of pHis1522 vector, carrying the xylose inducible promoter in Brevibacillus , allow a high level of intracellular protein expression. The yield obtained for GFP (250 mg/L) is more than 25 fold higher than that reported for B. megaterium carrying the same expression vector [26], more than 10-fold higher compared to that obtained with Brevibacillus carrying the GFP-pNI vector (Figure 5) based on the P2 constitutive promoter and, finally, comparable with E. coli in a fed-batch cultivation [27]. The yields obtained for both α-amylase and TcdA-GT using the pHis1522 vector were about 2-3 fold higher compared to those obtained using the pNI-His vector.…”

Section: Discussionmentioning

confidence: 93%

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

et al. 2013

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BackgroundIn past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus.ResultsUsing GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP) was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA). The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds) than those obtained using the available plasmids based on the P2 constitutive promoter.ConclusionCombining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio) and B. megaterium (from Mobitec), we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

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Identifying conditions for inducible protein production in E. coli: combining a fed-batch and multiple induction approach

Cited by 33 publications

References 20 publications

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

Analyzing the metabolic stress response of recombinant Escherichia coli cultures expressing human interferon-beta in high cell density fed batch cultures using time course transcriptomic data

Imaging bacterial protein expression using genetically encoded RNA sensors

High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

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