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E. coli Expression Strains

NEB offers several strains with varying levels of expression control, each with phage T1 resistance and extremely high transformation efficiencies.

NEB Express is an enhanced BL21 derivative available with (NEB #C3037) or without (NEB #C2523) the added control from lacIq.

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.

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Protocols for E. coli Expression Strains
Application Notes for E. coli Expression Strains
    Publications related to E. coli Expression Strains
    • Schlegel, S., Klepsch, M., Gialama, D., Wickström, D., Slotboom, D.J. and de Gier, J. (2010) Revolutionizing membrane protein overexpression in bacteria Microb Biotechnol; 3 , 403-411 .
    • Narayanan, A., Ridilla, M. and Yernool, D.A. (2010) Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator–repressor interactions Protein Sci; 20 , 51-61 .
    • 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), 992-1002. PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
    • 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), 1864-1869. PubMedID: 291309344​
    • Ren, G., Ke, N. and Berkmen, M. (2016) Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci; Aug 1, 1;85:5.26.1-5.26.21.. PubMedID: 27479507 , DOI: 10.1002/cpps.11.
    • 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, 101280. PubMedID: 31450103, DOI: 10.1016/j.redox.2019.101280
Advantages of SHuffle® Strains
  • 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
Advantages of NEB Competent Cells
  • 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
NEB Competent E. coli Strains
NEB offers several stains with varying levels of expression control, each with phage T1 resistance and extremely high transformation efficiencies.
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 a 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 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™).
Origami™ is a trademark of Invitrogen.
SHuffle® Strain Selection Chart
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