An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli.
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 sigma(32)-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.
Kraft
Mario
M
Department Pilot Plant for Natural Products, Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Beutenberg Strasse 11a, 07745, Jena, Germany.
Knüpfer
Uwe
U
Wenderoth
Rolf
R
Pietschmann
Patricia
P
Hock
Björn
B
Horn
Uwe
U
D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009862:Online Systems; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D012808:Sigma Factor / Q000378:metabolism*; D012995:Solubility
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D008156:Luciferases