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  • E. coli

    Protein Expression

    NEB offers two protein expression systems in E.coli. The pMAL™ Protein Fusion & Purification System (NEB #E8200) is used to express an MBP-fusion protein which is then purified by affinity purification. This system enhances solubility and results in reliable E.coli expression in either the cytoplasm or periplasm.

    The IMPACT™ Kit (NEB #E6901) allows fusion of a tag consisting of the intein and the chitin binding domain (CBD), to either the C-terminus (pTXB1) or the N-terminus (pTYB21) of the target protein. In the presence of thiols, such as DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in purification in a single chromatographic step with no need for proteases.

    pMAL™ and IMPACT™ are trademarks of New England Biolabs, Inc.

    E. coli includes these subcategories:

    IMPACT™
    pMAL™

    Schematic Illustration of Purification Using pMAL™ System

    Protein Expression Using pMAL™

    SDS-polyacrylamide gel electrophoresis of fractions from the purification of MBP-paramyosin-ΔSal.
    Lane 1: Protein Ladder (NEB #P7703).
    Lane 2: uninduced cells.
    Lane 3: induced cells.
    Lane 4: purified protein eluted from amylose column with maltose.
    Lane 5: purified protein after Factor Xa cleavage.
    Lane 6: paramyosin fragment in flow-through from second amylose column.

    Schematic Illustration of the IMPACT™ System

    Expression and Purification of E. coli Maltose-Binding Protein (MBP) Using the pTXB1 Vector (pMXB10)

    Lane 1: uninduced cell extract.
    Lane 2: induced cell extract showing expressed fusion protein.
    Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C.
    Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).

    T7-Controlled Expression of a Non-Toxic 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.

    Western Analysis of 6-His-tagged Brugia malayi Protein

    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).

    Overnight Expression of a Membrane Protein - PhoA fusion

    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 Activity Assayed from Crude Lysates

    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

    Purification workflow of target protein that has been expressed in the NiCo21(DE3) strain of E. coli.

    Optimization of YidC-GFP Expression with Lemo21(DE3)

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

    Lemo21(BE3) vs. BL21(DE3)

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