Expression and Fermentation Strategies for Recombinant Protein Production in Escherichia Coli

Neubauer, Peter; Winter, Jeannette

doi:10.1007/978-94-015-9749-4_17

Cited by 16 publications

(15 citation statements)

References 377 publications

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“…To obtain sufficient quantities of recombinant protein, the production was carried out in fermentors using a high cell density culture mode in a FedBatch process, where the addition of nutrients and growth ( ϭ 0.25 h Ϫ1 ) are automatically controlled. Several authors have already described that the overexpression of a recombinant gene induces a heat shock-like response that enhances both proteolytic activities and the levels of chaperones (35,36). Because YodA had been related to cellular stress, we first thought that the overexpression of a heterologous protein could trigger a cellular stress provoking YodA expression in E. coli.…”

Section: Resultsmentioning

confidence: 99%

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

David

Blondeau

Schiltz

et al. 2003

Journal of Biological Chemistry

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We have determined the crystal structure of YodA, an Escherichia coli protein of unknown function. YodA had been identified under conditions of cadmium stress, and we confirm that it binds metals such as cadmium and zinc. We have also found nickel bound in one of the crystal forms. YodA is composed of two domains: a main lipocalin/calycin-like domain and a helical domain. The principal metal-binding site lies on one side of the calycin domain, thus making YodA the first metal-binding lipocalin known. Our experiments suggest that YodA expression may be part of a more general stress response. From sequence analogy with the Cterminal domain of a metal-binding receptor of a member of bacterial ATP-binding cassette transporters, we propose a three-dimensional model for this receptor and suggest that YodA may have a receptor-type partner in E. coli.

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Section: Resultsmentioning

confidence: 99%

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

David

Blondeau

Schiltz

et al. 2003

Journal of Biological Chemistry

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“…4). It is known that the activity and the concentration of components of the cellular protein synthesis machinery show a direct relation to the actual growth rate (Neubauer and Winter, 2001), for instance, the number of ribosomes and the fraction of functioning ribosomes is increasing with increasing growth rate (Yun et al, 1991(Yun et al, , 1996. This suggests that, as a general rule, recombinant protein production is most efficient at higher growth rates.…”

Section: Discussionmentioning

confidence: 96%

Characterization of the AlkS/P_alkB‐expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed‐batch fermentations

Makart

Heinemann

Panke

2006

Biotech & Bioengineering

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The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P(alkB) expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single-cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed-back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned on at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations decreased from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose-limited fed-batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein.

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“…High synthesis rates may result in malfunction of the protein synthesis system of the host cell and a change of the central carbon metabolism (Dong et al, 1995;George et al, 1992;Jürgen et al, 2000;Rinas, 1996;Vind et al, 1993). Furthermore, overexpression of a recombinant gene can induce a heat-shock-like response, which enhances proteolytic activities and chaperone levels (for extensive review see Neubauer and Winter, 2001). The relation between induction of the stringent response and production of recombinant proteins has been investigated less frequently and has been more contradictory, which may be explained by the different routes of ppGpp synthesis in E. coli (relA and spoT routes).…”

Section: Introductionmentioning

confidence: 99%

“…The production of recombinant proteins can significantly influence cell metabolism by channeling bacterial resources toward the production of the target protein, thereby imposing a metabolic burden and stress to the host cell (for recent reviews see Hoffmann and Rinas, 2004;Neubauer and Winter, 2001). In particular, the strong synthesis of a recombinant protein can induce a number of different stress signals connected to cellular regulons such as a heat-shocklike or unfolded protein response (Dong et al, 1995;Jürgen et al, 2000;Parsell and Sauer, 1989), the stringent response (Cserjan-Puschmann et al, 1999), the general stress response (Schweder et al, 2002), and the SOS response (Arís et al, 1998;Lin et al, 2001), but it depends on the strength of competition for recombinant and stress protein production whether the corresponding gene expression pattern for the respective response is found ).…”

Section: Introductionmentioning

confidence: 99%

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high‐cell‐density cultures of Escherichia coli

Lin

Hoffmann

Rozkov

et al. 2004

Biotech & Bioengineering

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Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.

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Expression and Fermentation Strategies for Recombinant Protein Production in Escherichia Coli

Cited by 16 publications

References 377 publications

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

Characterization of the AlkS/P_alkB‐expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed‐batch fermentations

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high‐cell‐density cultures of Escherichia coli

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Expression and Fermentation Strategies for Recombinant Protein Production in Escherichia Coli

Cited by 16 publications

References 377 publications

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

YodA from Escherichia coli Is a Metal-binding, Lipocalin-like Protein

Characterization of the AlkS/PalkB‐expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed‐batch fermentations

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high‐cell‐density cultures of Escherichia coli

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Characterization of the AlkS/P_alkB‐expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed‐batch fermentations