Protein Expression
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  • E.coli Protein Expression

    Protein expression in the bacterium E. coli has been the most popular means of producing recombinant proteins for over two decades. E. coli is a well-established host that offers short culturing time, easy genetic manipulation and low cost media. Additionally, E. coli has a long history of being able to produce a wide variety of different types of proteins. Even proteins that contain disulfide bonds can be expressed within the cytoplasm of NEB SHuffle® strains.

    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. When inducer is added, T7 RNA polymerase is expressed and becomes dedicated to transcription of the gene of interest.

    SHuffle® is a registered trademark of New England Biolabs, Inc.

    Solutions for the Expression of Difficult Proteins

    NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.

    NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.

    FAQs for E.coli Protein Expression

    Protocols for E.coli Protein Expression

    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.

    Improvement of Protein Solubility with Lemo21(DE3)

    A) B. malayi protein expressed at 20°C in BL21(DE3).
    B) Soluble fractions of B. malayi protein expressed at 30°C in Lemo21 (DE3).

    The Lemo System™ for Membrane Protein Expression

    Lemo System™ enables simple, rapid optimization of membrane protein expression.

    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 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.

    Improved Purity of His-tagged Proteins with NiCo21(DE3)

    Expression of Glutamyl tRNA Synthetase (6-His) in NiCo21(DE3) Competent E. coli followed by Ni-NTA purification. Eluent (E) from a Ni-NTA column was passed over a chitin column. The protein of interest elutes in the flow through (FT), while the CBD-tagged metal binding proteins remain bound (B) to the chitin resin (NEB #S6651S). Purifications were performed according to manufacturers' recommended conditions. B) Contaminants ArnA, SlyD and Can are confirmed by Western blot using Anti-CBD Antibody (NEB #E8034S)

    NiCo21(DE3) Two-Step Purification

    Lemo21(DE3) achieves a higher level of expression than BL21(DE3) pLysS.

    Optimization of YidC-GFP Expression with Lemo21(DE3)

    Lemo21(DE3) achieves a higher level of expression than BL21(DE3) pLysS.

    Lemo21(DE3) vs BL21(DE3)

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