Abstract
BackgroundBacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield.ResultsHere, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts.ConclusionsThe plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.
Highlights
Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins
Its mode of regulation may differ among microbial species [5], in Gram-negative bacteria, ppGpp primarily interacts with RNA polymerase (RNAP), in synergy with the RNAP-binding transcriptional factor DksA, to directly affect transcription [6, 7]; and the ppGpp binding site regions are well conserved in representatives of the Gammaproteobacteria [8]
In Escherichia coli, overproduction of recombinant proteins can lead to formation of inclusion bodies if there is an imbalance between the rates of protein synthesis and folding
Summary
Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Analysis of the rpoH genes encoding homologs of σ32 protein from the Alpha- and Gammaproteobacteria subgroups revealed sequence similarities that should reflect conserved function and regulation of σ32 in the heatshock response [12]. These data are consistent with further observations that expression of σ32 homologs from Serratia marcescens, Proteus mirabilis and Pseudomonas aeruginosa in E. coli mutant strain lacking its own σ32 leads to the transcriptional activation of the heat-shock genes from the start sites normally used in E. coli [13]
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