Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E.coli.
Summary of "Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E.coli."
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.
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 Deltagor DeltatrxB strain rosetta-gami upon co-expression of Erv1p.
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.
This article was published in the following journal.
Name: Microbial cell factories
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/20836848
- DOI: http://dx.doi.org/10.1186/1475-2859-9-67
Medical and Biotech [MESH] Definitions
Sulfur-sulfur bond isomerases that catalyze the rearrangement of disulfide bonds within proteins during folding. Specific protein disulfide-isomerase isoenzymes also occur as subunits of PROCOLLAGEN-PROLINE DIOXYGENASE.
Protein synthesized by CLOSTRIDIUM TETANI as a single chain of ~150 kDa with 35% sequence identity to BOTULINUM TOXIN that is cleaved to a light and a heavy chain that are linked by a single disulfide bond. Tetanolysin is the hemolytic and tetanospasmin is the neurotoxic principle. The toxin causes disruption of the inhibitory mechanisms of the CNS, thus permitting uncontrolled nervous activity, leading to fatal CONVULSIONS.
A GLUTATHIONE dimer formed by a disulfide bond between the cysteine sulfhydryl side chains during the course of being oxidized.
Cystine Knot Motifs
Amino acid sequence in which two disulfide bonds (DISULFIDES) and their connecting backbone form a ring that is penetrated by a third disulfide bond. Members include CYCLOTIDES and agouti-related protein.
Hydrogen-donating proteins that participates in a variety of biochemical reactions including ribonucleotide reduction and reduction of PEROXIREDOXINS. Thioredoxin is oxidized from a dithiol to a disulfide when acting as a reducing cofactor. The disulfide form is then reduced by NADPH in a reaction catalyzed by THIOREDOXIN REDUCTASE.
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