Protein Expression
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  • Toxic Protein Expression

    Expression of heterologous proteins presents many challenges. In E. coli, expression of a non-native protein often adversely affects the viability of the host cell both during the transformation stage and during protein expression. To improve host viability and consequently improve the potential for target protein over-expression, well-regulated expression systems should be employed. In E. coli expression systems, regulation is often provided by the host cell as well as the expression vector. IPTG-inducible systems rely on the Lac repressor to bind to lac, tac, trc or T7-lac promoters to inhibit expression until the culture reaches an optimal density for induction. In T7 systems, the Lac repressor is also important but an even more effective means to control expression is to employ a host strain that expresses a T7 RNA polymerase inhibitor protein (e.g. LysY).

    • 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.

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    FAQs for Toxic Protein Expression

    Protocols for Toxic 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)

    1. B. malayi protein expressed at 20°C in BL21(DE3).
    2. 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.

    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™).

    Optimization of YidC-GFP Expression with Lemo21(DE3)

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

    Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit

    25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)

    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.