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.
FAQs for Disulfide-Bonded Protein Expression
- Does my protein have disulfide bonds?
- 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?
- What applications are SHuffle® strains useful for?
- Which SHuffle® strain should I use?
- How do SHuffle® strains aid in cytoplasmic disulfide bond formation?
Protocols for Disulfide-Bonded Protein Expression
- 5 Minute Transformation Protocol (C3026)
- 5 Minute Transformation Protocol (C3027)
- 5 Minute Transformation Protocol (C3028)
- 5 Minute Transformation Protocol (C3029)
- 5 Minute Transformation Protocol (C3030)
- 5 Minute Transformation Protocol (C3032)
- Affinity Purification and On-column Cleavage (E6901)
- Analysis of Synthesized Protein using PURExpress (E6800)
- Construction of the Fusion Plasmid (E6901)
- Determination of Protein Synthesis Yield with PURExpress (E6800)
- Expression Protocol (C3032)
- Expression Using SHuffle (C3026)
- Expression Using SHuffle (C3027)
- Expression Using SHuffle (C3028)
- Expression Using SHuffle (C3029)
- Expression Using SHuffle (C3030)
- Fusion Constructs (E6901)
- Fusion Protein Expression (E6901)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol
- Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
- Preparation of Media and Solutions (E6901)
- Primer Design for Restriction Enzyme Cloning (E6901)
- Protein Expression with T7 Strains
- Protein Synthesis Reaction using PURExpress (E6800)
- PURExpress Disulfide Bond Enhancer (E6820)
- Purification of Synthesized Protein using Reverse His-tag Purification
- Simplified Expression and Purification Protocol (E6901)
- Transformation Protocol (C3032)
Application Notes Disulfide-Bonded Protein Expression
Bypassing Common Obstacles in Protein Expression
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
- Competent Cell Product Comparison
- Convenient Formats of Competent Cells
Other Tools & Resources
Disulfide Bond Formation
PfCHT1 Chitinase Expression in Shuffle® T7 Express
vtPA Activity Expression in Shuffle®
What makes SHuffle cells so efficient at the expression of disulfide-bonded proteins?
What is a disulfide bond, and how are they formed?
Where, and under what conditions, can disulfide bonds form?
What are the steps of disulfide bond formation in the periplasm, and which proteins are responsible for successful bond formation?
NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.