NEB offers several strains with varying levels of expression control, each with phage T1 resistance and extremely high transformation efficiencies.
Within the realm of E. coli expression, the T7 system is the most popular approach for producing proteins. In this system, an expression vector containing a gene of interest cloned downstream of the T7 promoter is introduced into a T7 expression host. T7 expression hosts, such as DE3 strains or T7 Express strains, carry a chromosomal copy of the phage T7 RNA Polymerase gene, which is controlled by a lac promoter. When inducer is added, T7 RNA Polymerase is expressed and becomes dedicated to transcription of the gene of interest. T7 Express strains are available with the control features lacIq and/or lysY. The lysY gene expresses a variant T7 lysosome which naturally inhibits T7 RNA Polymerase, thereby controlling basal expression of the target gene. LysY is expressed from a single copy mini-F plasmid that does not require antibiotic selection for propagation. T7 Express lysY/Iq (NEB #C3013) provides the highest level of expression control.
BL21(DE3) (NEB #C2527) is available for routine T7 expression and Lemo21(DE3) (NEB #C2528) is designed for tunable T7 expression of difficult targets such as membrane proteins, toxic proteins and proteins prone to insoluble expression. NiCo21(DE3) (NEB #C2529) has been engineered to improve the recovery of His-tagged proteins.
SHuffle® strains enable disulfide bond formation in the cytoplasm. Normally, reductases in the E. coli cytoplasm keep cysteines in their reduced form, thereby preventing disulfide bond formation in this compartment. The deletions of the genes for glutaredoxin reductase and thioredoxin reductase (Δgor ΔtrxB), allows disulfide bond formation in the cytoplasm with SHuffle. This combination of mutations is normally lethal, but the lethality is suppressed by a mutation in the peroxiredoxin enzyme (ahpC*). In addition, SHuffle expresses a version of the periplasmic disulfide bond isomerase, DsbC, which lacks its signal sequence, retaining it in the cytoplasm. This enzyme has been shown to act on proteins with multiple disulfide bonds, to correct mis-oxidized bonds and promote proper folding.
- 5 Minute Transformation Protocol (C2527)
- 5 Minute Transformation Protocol (C2528)
- 5 Minute Transformation Protocol (C2529)
- 5 Minute Transformation Protocol (C2530)
- 5 Minute Transformation Protocol (C2566)
- 5 Minute Transformation Protocol (C3010)
- 5 Minute Transformation Protocol (C3013)
- 5 Minute Transformation Protocol (C3026)
- 5 Minute Transformation Protocol (C3028)
- 5 Minute Transformation Protocol (C3029)
- 5 Minute Transformation Protocol (C3030)
- 5 Minute Transformation Protocol (C3037)
- Expression Using SHuffle (C3026)
- Expression Using SHuffle (C3028)
- Expression Using SHuffle (C3029)
- Expression Using SHuffle (C3030)
- High Efficiency Transformation (C2523)
- High Efficiency Transformation Protocol (C2566)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C3013)
- High Efficiency Transformation Protocol
- High Efficiency Transformation Protocol (C3037)
- Protein Expression Using BL21(DE3) (C2527)
- Expression Using NEB Express (C2523)
- Protocol for Expression Using NEB Express Iq (C3037)
- Protein Expression Using NiCo21(DE3) (C2529)
- Protocol for Expression Using T7 Express (C2566)
- Protocol for Expression Using T7 Express lysY (C3010)
- Protocol for Expression Using T7 Express Crystal (C3022)
- Protocol for Expression Using T7 Express lysY/Iq (C3013)
- Protocol for Protein Expression Using BL21 (C2530)
- Protocol for Removal of IMAC Contaminating Proteins (C2529)
- Recommended media and expression conditions for T7 Express Crystal (C3022)
- Seleno-methionine Incorporation (C3022)
- Transformation Protocol (C2528)
- Transformation Protocol (C2530)
- Transformation Protocol for BL21(DE3) Competent Cells (C2527)
- High Efficiency Transformation Protocol (C2529)
- Protein Expression with T7 Express strains
- Expression Using SHuffle®
- Protein Expression Using Lemo21(DE3) (C2528)
- 5 Minute Transformation (C2523)
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
- Characteristics of Select E.coli Strains
- Competent Cell Product Comparison
- Competent Cell Selection Guide
- Troubleshooting Transformation Reactions
- Additional E. coli Strain Genotypes
- Electroporation Tips
- Genetic Markers
- Making Unmethylated (Dam- Dcm-) DNA
- McrA, McrBC and EcoKI Strain Phenotypes
- Restriction of Foreign DNA by E. coli K-12
- Reuter, W.H., Masuch, T., Ke, N., Lenon, M., Radzinski, M., Van Loi, V., Ren, G., Riggs, P., Antelmann, H., Reichmann, D., Leichert, L.I., Berkmen, M 2019. Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote Redox Biol. 26, PubMedID: 31450103, DOI: 10.1016/j.redox.2019.101280
- 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:
- 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
- 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:
- Hibender, S. Landeta, C., Berkmen, M., Beckwith, J., Boyd, D. 2017. Aeropyrum pernix membrane protein VKOR promotes protein disulfide bond formation in two subcellular compartments. Microbiology. 163 (12), PubMedID: 291309344, DOI:
- Ren, G., Ke, N. and Berkmen, M. 2016. Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci.. Aug 1, PubMedID: 27479507 , DOI: 10.1002/cpps.11.
- Oxidizing cytoplasmic environment enables disulfide bond formation
- DsbC (disulfide bond isomerase) directs correct disulfide bond formation
- DsbC also acts as a general chaperone for protein folding
- Cytoplasmic expression increases protein yield
- A wide range of antibiotics can be used for plasmid maintenance (Amp, Kan, Tet, Cam [except for lysY versions])
- Transformation efficiency: 1 x 106 cfu/µg pUC19 DNA
- Protease deficient
- TI phage resistant (fhuA2)
- Free of animal products
- Free of animal products
- T1 phage resistance (fhuA2)
- Media and control plasmid included with most strains
- A variety of convenient formats, including single use transformation tubes and, on request, 96 well plates
- Bulk sales capabilities with custom packaging
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In this video, see how transformation plating can be fast and easy using glass beads.