Protein Expression
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  • Expression of Difficult Proteins

    Many proteins are extremely difficult to express in heterologous expression systems. A vast number of factors may contribute to this problem. A common problem is that it can often be challenging for a foreign host to correctly fold a protein it does not normally produce. For example, in many experimental scenarios expression of a protein originating from a higher eukaryote is being produced in a bacterium where factors such as codon usage, translation rate, and redox potential are significantly different. Additionally, inherent properties of the target protein may represent challenges for the expression host. For example, a protein having multiple membrane spanning domains might not properly insert into membrane bilayers of the heterologous host or a protein might not be expressed in a soluble form. Finally, many proteins require post-translational modifications (e.g. glycosylation or phosphorylation) that are absent or significantly different from expression host to expression host.

    There is no single solution for the expression of all classes of difficult proteins. Instead, expression problem-specific solutions that aim to better the chances of success can be used. For microbial expression systems, these solutions often come in the form of unique host strains that have been genetically modified to enhance the production of a certain difficult protein class. Other expression solutions seek to address problems by controlling aspects of how a target protein is produced. For example, some expression hosts allow for precise control of target gene expression. In addition, certain protein tags can help a protein to more efficiently insert into a host membrane or improve the solubility of a target protein.

    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.

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    Expression of Difficult Proteins includes these areas of focus:

    Toxic Protein Expression
    Disulfide-Bonded Protein Expression
    Membrane Protein Expression

    FAQs for Expression of Difficult Proteins

    Protocols for Expression of Difficult Proteins

    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.

    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™).

    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.

    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.