The redox state of the cytoplasm of eukaryotic and prokaryotic cells is reducing, due to the presence of numerous disulfide bond reductases, such as 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 compartmentalized outside of the reducing cytoplasm. [see animation 2 of 4, at right] In eukaryotes, disulfide bond formation is catalyzed by Protein Disulfide Bond Isomerase (PDI) in the endoplasmic reticulum (ER), whereas in prokaryotes, disulfide bond formation is catalyzed by DsbA in the periplasm. [see animation 3 of 4, at right] 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.
There are several options for expressing proteins that require disulfide bonding for proper folding and function. The E. coli strain SHuffle® has been genetically engineered for the cytoplasmic production of disulfide-bonded proteins. Genetic deletion of the two (glutaredoxin and thioredoxin) reducing pathways in E. coli has resulted in a mutant strain with diminished capacity to reduce proteins, and an increased capacity to oxidize cytoplasmically-expressed proteins. Additionally, these cells have been engineered to express the disulfide bond isomerase DsbC in the cytoplasm, further enhancing the capacity for and fidelity of disulfide bond formation in this host. [see animation 4 of 4, at right]
|Strain||NEB #||Characteristics||Drug Resistance|
|SHuffle Express Competent E. coli||C3028||
|SHuffle T7 Express Competent E. coli||C3029||
|SHuffle T7 Express lysY Competent E. coli||C3030||
|SHuffle T7 Competent E. coli||C3026||
* Resistance to low levels of steptomycin may be observed.It is also possible to use eukaryotic protein secretion or cell-free expression strategies to achieve proper disulfide bonding of proteins. For example, secretion of a target protein into the oxidative ER environment of yeast, mammalian or insect cells will permit a nascent protein the opportunity to fold in and to have access to PDI. Cell-free strategies can also be employed using systems like PURExpress® with Disulfide Bond Enhancer that contains components that improve in vitro formation of disulfide bonds.
Protocols for SHuffle® strains for the expression of multi-disulfide bonded and difficult-to-express proteins
- Protein Expression Using BL21(DE3) (C2527)
- Protocol for Removal of IMAC Contaminating Proteins (C2529)
- Expression Using NiCo21(DE3) (C2529)
- Protein Expression Using Lemo21(DE3) (C2528)
- Protocol for Expression Using T7 Express Crystal (C3022)
- Protocol for Protein Expression Using BL21 (C2530)
- Protocol for Expression Using T7 Express Iq (C3037)
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express lysY (C3010)
- Expression Using NEB Express (C2523)
- Transformation Protocol (C2528)
- Transformation Protocol (C2530)
- Recommended media and expression conditions for T7 Express Crystal (C3022)
- Seleno-methionine Incorporation (C3022)
- 5 Minute Transformation Protocol (C3029)
- 5 Minute Transformation Protocol (C2566)
- 5 Minute Transformation Protocol (C3026)
- 5 Minute Transformation Protocol (C3028)
- 5 Minute Transformation Protocol (C3037)
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C2529)
- 5 Minute Transformation Protocol (C3010)
- 5 Minute Transformation Protocol (C3022)
- 5 Minute Transformation Protocol (C2530)
- 5 Minute Transformation Protocol (C3030)
- Expression Using SHuffle (C3026)
- Expression Using SHuffle (C3030)
- Expression Using SHuffle (C3028)
- Expression Using SHuffle (C3029)
- High Efficiency Transformation Protocol (C3037)
- High Efficiency Transformation (C2523)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C3022)
- High Efficiency Transformation Protocol (C2566)
- High Efficiency Transformation Protocol (C2529)
- 5 Minute Transformation (C2523)
- High Efficiency Transformation Protocol (C3013)
- 5 Minute Transformation Protocol (C3013)
- Protocol for Expression Using T7 Express lys/Iq(C3013)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
- 5 Minute Transformation Protocol (C2527)
- Protein Expression with T7 Strains
- Expression Using SHuffle®
- E. coli Lemo21(DE3)
- Protein Expression with T7 Express Strains
- Transformation of SHuffle® Competent Cell Strains
Competent Cells Brochure
The Competent Cells brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
Protein Expression & Purification Brochure
The Protein Expression and Purification brochure provides information on the advanced tools for protein expression and purification offered by NEB.
Bypassing Common Obstacles in Protein Expression
- Characteristics of Select E.coli Strains
- Competent Cell Product Comparison
Competent Cell Selection Guide
- Strain Properties
- Troubleshooting Transformation Reactions
- Additional E. coli Strain Genotypes
- Convenient Formats of Competent Cells
- Electroporation Tips
- Genetic Markers
- Making Unmethylated (Dam- Dcm-) DNA
- McrA, McrBC and EcoKI Strain Phenotypes
- Restriction of Foreign DNA by E. coli K-12
Other Tools & Resources
- Narayanan, A., Ridilla, M. and Yernool, D.A. 2010. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operatorâ€“repressor interactions Pro. Sci. . 20 , PubMedID: , DOI:
- Schlegel, S., Klepsch, M., Gialama, D., Wickström, D., Slotboom, D.J. and de Gier, J. 2010. Revolutionizing membrane protein overexpression in bacteria Micro. Biotech. . 3 , PubMedID: , DOI:
- Wagner, S., Klepsch, M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Hö, M., van Wijk, K.J., Slotboom, D.J., Persson, J.O. and de Gier, J. 2008. Tuning Escherichia coli for membrane protein overexpression PNAS . 105 , PubMedID: , DOI:
Publications related to SHuffle® strains for the expression of multi-disulfide bonded and difficult-to-express proteins
DNA Polymerase Selection Chart
Template/product specificity: Is RNA or DNA involved? Is the 3´ terminus at a gap, nick or at the end of the template?
Removal of existing nucleotides: Will the nucleotide(s) be removed from the existing polynucleotide chain as part of the protocol? If so, will they be removed from the 5´ or the 3´ end?
Thermal stability: Does the polymerase need to survive incubation at high temperature or is heat inactivation desirable?
Fidelity: Will subsequent sequence analysis or expression depend on the fidelity of the synthesized products?
PCR Selection Tool
Taq Buffer Selection Chart
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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?