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.

Recombinant protein production associated growth inhibition results mainly from transcription and not from translation

“…This is presumably the case for hFGF-2. hFGF-2 is a slow folding protein [20] that readily forms inclusion bodies and elicits a strong heat-shock response during rapid production in batch cultures [16] this way most likely increasing the transcription related growth inhibition.…”

“…1). For example, the production of hFGF-2, a protein partly produced as soluble protein and partly in form of inclusion bodies, leads to a strong impact on bacterial growth ( [14][15][16], Fig. 1a).…”

Potential Applications of the Escherichia coli Heat Shock Response in Synthetic Biology

“…HSP genes, such as those encoding chaperones or other gene products that can increase the HSR when expressed (e.g., rpoH), can also be very useful parts in synthetic biology given their potential role in heterologous protein folding [5] (see the 'Industrial Applications' section).…”

“…However, many eukaryotic proteins of interest such as therapeutic proteins (e.g., antibodies) have complex tertiary or quaternary structures that usually require post-translational modifications such as glycosylation, phosphorylation, and addition of fatty acid chains that are necessary to obtain correct folding and biological activity [29]. Therefore, the overexpression of these heterologous proteins often causes an imbalance in cell equilibrium that results in misfolding and aggregation of recombinant proteins as inclusion bodies [5,46]. The increase of misfolded proteins and aggregates induces the E. coli HSR.…”

“…The HSR is an essential mechanism to protect cells against adverse conditions including heat shock (HS), viral infection [1], UV irradiation [2], high alcohol concentrations [1], heavy metals [3], oxidative stress [4], and recombinant protein production [5]. These stress factors increase the levels of denatured proteins which can be harmful to the cell owing to changes in their biological activities.…”