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  • SHuffle® Sampler Pack

    Description

    SHuffle® strains from NEB are engineered E. coli strains capable of expressing proteins with increasing disulfide bond complexity in the cytoplasm. SHuffle strains express the disulfide bond isomerase DsbC within the cytoplasm. DsbC isomerizes mis-oxidized substrates into their correctly folded state greatly enhancing the fidelity of disulfide bond formation. Cytoplasmic expression also results in significantly higher protein yields of disulfide bonded proteins when compared to periplasmic expression. SHuffle strains are sensitive to kan, amp, tet and in most cases, cam, which makes them able to express proteins from a wide variety of expression vectors offering greater versatility in experimental design.

    SHuffle strains can correctly fold disulfide bonds in the cytoplasm
    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.

    Figure 1: Express higher levels of biologically active protein with 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™).

    Figure 2: SHuffle offers robustness and less toxicity than origami strains. 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.

    *Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.

    Highlights

    • Oxidizing cytoplasmic environment enables disulfide bond formation
    • DsbC (disulfide bond isomerase) directs correct disulfide bond formation
    • DsbC also acts as a chaperone for protein folding
    • Cytoplasmic expression increases protein yield of disulfide bonded proteins
    • A wide range of antibiotics can be used for plasmid maintenance (Amps, Kans, Tets, Cams [except for lysY versions])
    • Transformation efficiency: 
    • 1 x 106 cfu/µg pUC19 DNA (SHuffle)
    • 1 x 107 cfu/µg pUC19 DNA (SHuffle Express)
    • Protease deficient
    • TI phage resistant (fhuA2)
    • Free of animal products

    Genotype

    MiniF lacIq (CamR) / fhuA2 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA Δ(mcrC-mrr)114::IS10

    Properties and Usage

    Antibiotics for Plasmid SelectionWorking Concentration
    Ampicillin100 μg/ml
    Carbenicillin100 μg/ml
    Chloramphenicol33 μg/ml
    Kanamycin30 μg/ml
    Streptomycin25 μg/ml
    Tetracycline15 μg/ml

    Storage Temperature

    -80°C

    References

    1. Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
    2. Qiu, J., Swartz, J.R. and Georgiou, G. (1998). Appl. Environ. Microbiol.. 64, 4891-4896.
    3. Levy, R. et al. (2001). Protein Expr. Purif.. 23, 338-347.
    4. Chen, J. et al. (1999). J. Biol. Chem.. 274, 19601-19605.
    5. Boyd, D. et al. (2000). J. Bacteriol.. 182, 842-847.
    6. de Marco, A. (2009). Microbial Cell Factories. 8, 26.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].
    1. What applications are SHuffle® strains useful for?
    2. Which SHuffle® strain should I use?
    3. Is there anything I can do to increase protein yield when using SHuffle strains?
    4. What are the growth characteristics of the SHuffle strains?
    5. How do SHuffle® strains aid in cytoplasmic disulfide bond formation?
    6. Can I store competent cells at -20°C instead of -80°C?
    7. How should I express my protein of interest in SHuffle?
    8. Do Streptomycin and/or Spectinomycin need to be added to SHuffle strains?
    9. Which SHuffle strains are resistant to chloramphenicol? Is chloramphenicol required for maintenance of the mini F plasmid?
    10. Do SHuffle cells grow in minimal media?
    11. Are SHuffle strains temperature sensitive?
    1. 5 Minute Transformation Protocol (C3032)
    2. Expression Protocol (C3032)
    3. Transformation Protocol (C3032)

    Selection Tools

    Usage Guidelines & Tips