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.
- Does my protein have disulfide bonds?
- What applications are SHuffle® strains useful for?
- Which SHuffle® strain should I use?
- How do SHuffle® strains aid in cytoplasmic disulfide bond formation?
- Is the PURExpress® In Vitro Protein Synthesis Kit capable of dealing with disulfide bonds? If not, can you recommend something else to use post synthesis?
- Analysis of Synthesized Protein using PURExpress (E6800)
- Determination of Protein Synthesis Yield with PURExpress (E6800)
- Expression Using SHuffle (C3026)
- Expression Using SHuffle (C3028)
- Expression Using SHuffle (C3029)
- Expression Using SHuffle (C3030)
- High Efficiency Transformation Protocol
- Protein Synthesis Reaction using PURExpress (E6800)
- PURExpress Disulfide Bond Enhancer (E6820)
- Protein Expression with T7 Express strains
- Purification of Synthesized Protein using Reverse His-tag Purification
- Preparation of Media and Solutions (E6901)
- High Efficiency Transformation Protocol
- Transformation Protocol (C3032)
- 5 Minute Transformation Protocol (C3026)
- Fusion Constructs (E6901)
- Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
- 5 Minute Transformation Protocol (C3028)
- Simplified Expression and Purification Protocol (E6901)
- Expression Protocol (C3032)
- Construction of the Fusion Plasmid (E6901)
- 5 Minute Transformation Protocol (C3030)
- 5 Minute Transformation Protocol (C3032)
- Affinity Purification and On-column Cleavage (E6901)
- 5 Minute Transformation Protocol (C3027)
- 5 Minute Transformation Protocol (C3029)
- Fusion Protein Expression (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Expression Using SHuffle (C3027)
Avoid Common Obstacles in Protein Expression
Read how to avoid common obstacles in protein expression that prevent interactions with cellular machinery.
- Competent Cell Brochure
- Protein Expression & Purification Brochure
- Competent Cell Product Comparison
- Competent Cell Selection Guide
- Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkmen, 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 Expression and Purification. 153, PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
- 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 Journal of Biological Chemistry. 294(48), PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
- 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
- Ke, Na; Berkmen, Mehmet; Ren, Guoping; 2017. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nature Chemical Biology. 13, PubMedID: 28628094, DOI: 10.1038/nchembio.2409
- 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 Nature Communications . (6)8072, PubMedID: , DOI: 10.1038/ncomms9072.
- 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 Antioxidant and Redox Signaling. , PubMedID: 26191605, DOI: 10.1089/ars.2014.6235
- Berkmen, M. 2012. Production of disulfide-bonded proteins in Escherichia coli Protein Expression and Purification. , PubMedID: 22085722, DOI:
- 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:
- Manta, Bruno; Berkmen, Mehmet; . Disulfide Bond Formation in the Periplasm of Escherichia coli EcoSal Plus. , PubMedID: , DOI: 10.1128/ecosalplus.ESP-0012-2018.
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What makes SHuffle cells so efficient at the expression of disulfide-bonded proteins?