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Global proteome response of Escherichia coli BL21 to production of human basic fibroblast growth factor in complex and defined medium.

ABSTRACT: The global proteome response toward recombinant protein production in Escherichia coli BL21 (DE3) grown in complex and defined medium was analyzed. Overproduction of human basic fibroblast growth factor (hFGF-2), a difficult-to-fold protein, led to a reconstruction of the bacterial proteome. For example, heat shock chaperones were highly upregulated, especially when production occurred during fast growth in complex medium. Although heat shock chaperones increased to higher levels in complex medium more hFGF-2 accumulated within inclusion bodies indicating that the capacity to chaperone protein folding was not sufficient for high speed production. In both types of media, cellular proteins from substrate transport systems, central metabolic pathways, and by-product uptake (e.g. acetate) were downregulated. This downregulation was connected to growth inhibition and metabolic perturbations. For example, during production in complex and defined medium acetate reassimilation and glucose uptake, respectively, were severely hampered. Cellular proteins for degradation of less favorable substrates, elimination of reactive oxygen species, and DNA protection were also downregulated in response to hFGF-2 production. The decrease of proteins involved in transport, central metabolic pathways, and general cell protection was more pronounced in the fast producing culture in complex medium than in the slow producing culture in defined medium. In general, production of hFGF-2 seems to interfere with the adaptation process to changing growth conditions, in this case the adaptation from exponential growth to stationary phase.

SUBMITTER: Li Z

PROVIDER: S-EPMC6999367 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Global proteome response of <i>Escherichia coli</i> BL21 to production of human basic fibroblast growth factor in complex and defined medium.

Li Zhaopeng Z Nimtz Manfred M Rinas Ursula U

Engineering in life sciences 20170620 8

The global proteome response toward recombinant protein production in <i>Escherichia coli</i> BL21 (DE3) grown in complex and defined medium was analyzed. Overproduction of human basic fibroblast growth factor (hFGF-2), a difficult-to-fold protein, led to a reconstruction of the bacterial proteome. For example, heat shock chaperones were highly upregulated, especially when production occurred during fast growth in complex medium. Although heat shock chaperones increased to higher levels in compl ...[more]

PMID: 32624836

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Project description:BackgroundThe proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis.ResultsThe presence of preformed biomass precursor molecules in rich media such as Luria Bertani supported rapid growth concomitant to acetate formation and apparently unbalanced abundances of central metabolic pathway enzymes, e.g. high levels of lower glycolytic pathway enzymes as well as pyruvate dehydrogenase, and low levels of TCA cycle and high levels of the acetate forming enzymes Pta and AckA. The proteome of cells growing exponentially in glucose-supplemented mineral salt medium was dominated by enzymes of amino acid synthesis pathways, contained more balanced abundances of central metabolic pathway enzymes, and a lower portion of ribosomal and other translational proteins. Entry into stationary phase led to a reconstruction of the bacterial proteome by increasing e.g. the portion of proteins required for scavenging rare nutrients and general cell protection. This proteomic reconstruction during entry into stationary phase was more noticeable in cells growing in rich medium as they have a greater reservoir of recyclable proteins from the translational machinery.ConclusionsThe proteomic comparison of cells growing exponentially in different media reflected the antagonistic and competitive regulation of central metabolic pathways through the global transcriptional regulators Cra, Crp, and ArcA. For example, the proteome of cells growing exponentially in rich medium was consistent with a dominating role of phosphorylated ArcA most likely a result from limitations in reoxidizing reduced quinones in the respiratory chain under these growth conditions. The proteomic alterations of exponentially growing cells into stationary phase cells were consistent with stringent-like and stationary phase responses and a dominating control through DksA-ppGpp and RpoS.

Dataset Information

Global proteome response of Escherichia coli BL21 to production of human basic fibroblast growth factor in complex and defined medium.

Publications

Global proteome response of <i>Escherichia coli</i> BL21 to production of human basic fibroblast growth factor in complex and defined medium.

Similar Datasets

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