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An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli

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Abstract

The expression of heterologous proteins in the cytoplasm of Escherichia coli is often accompanied by limitations resulting in uncontrollable fermentation processes, increased rates of cell lysis, and thus limited yields of target protein. To deal with these problems, reporter tools are required to improve the folding properties of recombinant protein. In this work, the well-known σ32-dependent promoters ibpAB and fxsA were linked in a tandem promoter (ibpfxs), fused with the luciferase reporter gene lucA to allow enhanced monitoring of the formation of misfolded proteins and their aggregates in E. coli cells. Overexpression of MalE31, a folding-defective variant of the maltose-binding protein, and other partially insoluble heterologous proteins showed that the lucA reporter gene was activated in the presence of these misfolded proteins. Contrary to this, the absence of damaged proteins or overexpression of mostly soluble proteins led to a reduced level of luciferase induction. Through performing expression of aggregation-prone proteins, we were able to demonstrate that the ibpfxs::lucA reporter unit is 2.5–4.5 times stronger than the single reporter units ibp::lucA and fxs::lucA. Data of misfolding studies showed that this reporter system provides an adequate tool for in vivo folding studies in E. coli from microtiter up to fermentation scales.

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Acknowledgements

Thanks to Gisela Sudermann for excellent technical support. Special thanks to Giles Johnson and Friedrich Kring for critical reading of the manuscript. This work was funded by Merck KGaA Darmstadt, Germany.

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Correspondence to Uwe Horn.

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Kraft, M., Knüpfer, U., Wenderoth, R. et al. An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli . Appl Microbiol Biotechnol 75, 397–406 (2007). https://doi.org/10.1007/s00253-006-0815-6

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Keywords

  • Green Fluorescent Protein
  • Luciferase Activity
  • Heterologous Protein
  • Heat Shock Response
  • Insoluble Protein