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Disulfide-bonded Protein Expression

Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. Disulfide bonds can greatly increase the stability of a protein and are primarily found in proteins that reside outside the chaperone rich protective environment of the cytoplasm (e.g. secreted peptides, hormones, antibodies, interferons, extracellular enzymes, etc). Disulfide bonds can also serve catalytic (e.g. oxidoreductases) and signaling roles (e.g. oxidative stress response).

The redox state of the cytoplasm of eukaryotic and prokaryotic cells is reducing due to the presence of numerous disulfide bond reductases (e.g. thioredoxins and glutaredoxins). As such, any disulfide bond formed between two cysteines will quickly and efficiently be reduced back to its thiolate state. To form stable disulfide bonds within proteins, disulfide bond formation is typically segregated to compartments outside of the reducing cytoplasm. In eukaryotes, disulfide bond formation is catalyzed by protein disulfide bond isomerase (PDI) in the endoplasmic reticulum (ER), whereas in prokaryotes it is catalyzed by DsbA in the periplasm. An inherent problem in the process of disulfide bond formation is mis-pairing (mis-oxidation) of cysteines, which can cause misfolding, aggregation and ultimately result in low yields during protein production. Proteins that are mis-oxidized must be repaired and disulfide bonds must be shuffled back to their correctly oxidized native state. This is achieved by PDI in eukaryotes and DsbC by prokaryotes.

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    Publications related to Disulfide-bonded Protein Expression
  1. Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkm​en, M., Ke, N., Colussi, P. 2019. Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli. Protein Expr Purif. 153, PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
  2. Leith, E.M., O'Dell, W.B., Ke, N., McClung, C., Berkmen, M., Bergonzo, C., Brinson, R.G., Kelman, Z 2019. Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli J Biol Chem. 294(48), PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
  3. Reddy, P.T., Brinson, R.G., Hoopes, J.T., McClung, C., Ke, N., Kashi, L. 2018. Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli. mAbs MAbs. 10 (7), PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
  4. Ke, Na; Berkmen, Mehmet; Ren, Guoping; 2017. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nat Chem Biol. 13, PubMedID: 28628094, DOI: 10.1038/nchembio.2409
  5. Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T.J., Tuckey, C., Riggs, P.D., Colussi, P.A., Noren, C.J., Taron, C.H., Delisa, M.P., Berkmen, M. 2015. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria Nat Commun. (6)8072, PubMedID: , DOI: 10.1038/ncomms9072.
  6. Chatelle C, Kraemer S, Ren G, Chmura H, Marechal N, Boyd D, Roggemans C, Ke N, Riggs P, Bardwell J, Berkmen M 2015. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase Antioxid Redox Signal. , PubMedID: 26191605, DOI: 10.1089/ars.2014.6235
  7. Berkmen, M. 2012. Production of disulfide-bonded proteins in Escherichia coli Protein Expr Purif. , PubMedID: 22085722, DOI:
  8. Shouldice, S.R., Cho, S.H., Boyd, D., Heras, B., Eser, M., Beckwith, J., Riggs, P., Martin, J.L.and Berkmen, M. 2010. In vivo oxidative protein folding can be facilitated by oxidation-reduction cycling. Mol Microbiol. 75(1), PubMedID: 19968787, DOI:
  9. Manta, Bruno; Berkmen, Mehmet; . Disulfide Bond Formation in the Periplasm of Escherichia coli EcoSal Plus. , PubMedID: , DOI: 10.1128/ecosalplus.ESP-0012-2018.
Disulfide Bond Formation
Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle® is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.
PfCHT1 Chitinase Expression in Shuffle® T7 Express
Plasmodium falciparum chitinase (PfCHT1) with three cysteines was expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.
vtPA Activity Expression in Shuffle®
Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt + cells are DHB4, which is the parent of FÅ113 (Origami™).
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Videos

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    Disulfide bond formation in the cytoplasm of SHUFFLE® cells (4 of 4)

    What makes SHuffle cells so efficient at the expression of disulfide-bonded proteins?

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