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T7 Expression

Within the realm of E. coli expression, the T7 system is the most popular approach for producing recombinant protein. In this system, the target gene is cloned into an expression vector downstream of the T7 promoter and this construct is introduced into a T7 expression host. NEB offers several T7 system strains of competent E. coli for protein expression.

  • BL21(DE3) Competent E. coli (NEB #C2527), are chemically competent E. coli cells suitable for transformation and routine protein expression. This strain expresses the T7 RNA polymerase and is deficient in proteases Lon and OmpT. These cells are extremely pure and free of animal products.
  • Lemo21(DE3) Competent E. coli (NEB #C2528) is a BL21(DE3) strain containing the Lemo System™, offering tunable expression of difficult clones. Tunable expression is achieved by varying the level of lysozyme (lysY), the natural inhibitor of T7 RNA polymerase. The level of lysozyme is modulated by adding L-rhamnose to the expression culture at levels from zero to 2000 µM. When Lemo21(DE3) is grown without rhamnose, the strain performs the same as a pLysS containing strain. However, optional addition of rhamnose tunes the expression of the protein of interest. For difficult soluble proteins, tuning the expression level may also result in more soluble, properly folded protein.
  • NiCo21(DE3) Competent E. coli (NEB #C2529) are chemically competent E. coli cells derived from BL21(DE3). Poly-histidine tagged recombinant proteins that are isolated by immobilized metal affinity chromatography (IMAC) are often contaminated with significant amounts of endogenous E. coli metal binding proteins. The protein expression strain NiCo21(DE3) has been engineered to minimize E. coli protein contamination of IMAC fractions: endogenous GlmS is mutated to eliminate binding to IMAC resins and three other proteins (SlyD, ArnA and Can) are tagged to enable rapid removal by chitin affinity chromatography.
  • The T7 Express High Efficiency Group of Competent Cells includes three different strains, including T7 Express lysY/Iq Competent E. coli (High Efficiency) (NEB #C3013), T7 Express lysY Competent E. coli (High Efficiency) (NEB #C3010) and T7 Express Competent E. coli (High Efficiency) (NEB #C2566).

 

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FAQs for T7 Expression
Protocols for T7 Expression
Application Notes for T7 Expression
NiCo21(DE3) outperforms BL21(DE3) for His-tagged protein purity
 
His-tagged Glutamyl-tRNA synthetase (GluRS) was expressed in BL21(DE3) and NiCo21(DE3) under identical conditions, followed by target protein isolation by Ni-NTA resin (Qiagen). Ni-NTA elution fractions (E) have different E. coli contaminants because three essential contaminant proteins are tagged with chitin binding domain (CBD) within NiCo21(DE3). The NiCo21(DE3) elution was incubated with chitin resin, improving GluRS purity to 97% (FTc). Protein purity was assessed using a LabChip GXII system (Perkin Elmer). M= ColorPlus prestained protein ladder; L = Ni-NTA Load; ft = Ni-NTA flow-through; E = Ni-NTA elution, FTc = chitin column flow-through. Reference: Robichon et al. (2011) Appl. Environ. Microbiol. 77:4634-4646.
T7 Controlled Expression of Protein in
E. coli Hosts
A T7 expression plasmid containing a gene encoding E. coli UDG was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of expression in the T7 Express hosts while maintaining high levels of induced expression.
Lemo21(BE3) vs. BL21(DE3)

Protein expression with Lemo21(DE3) is very similar to BL21(DE3), with only a few minor changes.

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