title: Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.
abstract: BACKGROUND
  The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction.
  
  
  RESULTS
  Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p.
  
  
  CONCLUSIONS
  Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.
authors.affiliation: Department of Biochemistry, Linnanmaa Campus, University of Oulu, 90570 Oulu, Finland.
authors.forename: Feras
authors.initials: F
authors.lastname: Hatahet
authors.forename: Van Dat
authors.initials: VD
authors.lastname: Nguyen
authors.forename: Kirsi E H
authors.initials: KE
authors.lastname: Salo
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ruddock
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000378:metabolism; D000135:Acid Phosphatase / Q000737:chemistry / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D010832:6-Phytase; D000135:Acid Phosphatase; D000469:Alkaline Phosphatase; D004220:Disulfides; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C114089:phoA protein, E coli; C406788:appA protein, E coli