Protein Expression
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  • Membrane Protein Expression

    Integral membrane proteins constitute a significant fraction of the proteome of all living cells. Important examples of this protein class include certain signal transduction receptors, cell adhesion molecules and ion channels. The biochemical and structural characterization of this challenging class of protein represents an important frontier in both basic science and drug discovery research. However, due to their unique physical properties and requirement for association with cellular membranes, expression of membrane proteins in heterologous systems is often daunting.

    Cellular expression of recombinant membrane protein often results in protein aggregation and misfolding due to the hydrophobic nature of transmembrane segments. When using E. coli as a host, it is advantageous to express membrane proteins in moderation to avoid saturation of the membrane protein biogenesis pathway, which may lead to cell death and/or inclusion body formation. The Lemo21(DE3) protein production strain was designed for tunable T7 expression to achieve optimal assembly of transmembrane proteins or the optimal folding of soluble proteins. In standard T7 expression strains (e.g. BL21(DE3)) transcription of a target gene from a T7 promoter may be too robust and it is not easily controlled. In contrast, Lemo21(DE3) expresses a T7 RNA polymerase inhibitor protein (LysY) so that the level of target gene transcription may be precisely regulated. Lemo21(DE3) allows researchers to sample a wide range of expression levels to find the optimal conditions for each unique target protein. In the case of membrane proteins less expression often results in more functional 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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    FAQs for Membrane Protein Expression

    Protocols for Membrane 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.

    Optimization of YidC-GFP Expression with Lemo21(DE3)

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