E. coli is a common expression host in both research and industry, however the reducing environment of the cytoplasm creates challenges for soluble expression of proteins with cysteine residues. Approximately 50% of cysteine residues in heterologous proteins form disulfide bonds required for proper folding and activity. These disulfide bonds are reduced in the E. coli cytoplasm resulting in misfolded inactive protein and aggregation in inclusion bodies. Previously strains have been engineered to constitutively increase the oxidative potential of E. coli’s cytoplasm by deleting key enzymes in reducing pathways including glutathione oxidoreductase (gor) and thioredoxin reductase (trxB). However, constitutive oxidative stress has a toxic effect, slowing growth and limiting conditions for expression. Additionally, overexpression of a disulfide bond isomerase (dsbC) and disulfide bond oxidase (evr1p) have been shown to improve soluble expression of proteins containing disulfide bonds. Specifically, evr1p catalyzes cysteine oxidation and dsbC isomerizes disulfide bonds to improve correct folding when multiple disulfide bonds are present. We report improved cytoplasmic expression of disulfide containing recombinant proteins in engineered E. coli with two-stage dynamic control over the redox state, disulfide oxidase and isomerase activities. Recombinant proteins are expressed in a phosphate limited stationary phase coincident with dynamic control over cytoplasmic reducing power. An oxidized environment is created in the cytosol by deleting gor and dynamic reductions trxB and glutamate-cysteine ligase (gshA) levels upon entry into stationary phase. Additionally, increases in correctly folded disulfide bonds are obtained through overexpression of dsbC and evr1p, again upon entry into stationary phase. Tightly controlled expression and a reducing cytoplasm prior to phosphate depletion enables robust exponential growth and autoinduction of both an oxidative environment and heterologous protein expression upon phosphate depletion. Disulfide containing proteins with improved expression include single chain variable fragments, human hyaluronidase-I, and tissue plasminogen activator which contain 3-17 disulfide bonds. The host strains and plasmids offer a tightly controlled, robust and scalable approach for the expression and purification of disulfide containing proteins.

Two-stage dynamic control over redox state improves cytosolic expression of disulfide containing proteins in E. coli

ACS Spring Meeting 2021

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