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  • pMAL™ Protein Fusion and Purification System

    Description

    • Reliable expression: substantial yields (up to 100 mg/L) in more than 75% of the cases tested
    • Expression in either the cytoplasm or periplasm: periplasmic expression enhances folding of proteins with disulfide bonds
    • Fusion to MBP has been shown to enhance the solubility of proteins expressed in E. coli (6)
    • Gentle elution with maltose: no detergents or harsh denaturants

    In the pMAL™ Protein Fusion and Purification System, the cloned gene is inserted into a pMAL  vector down-stream from the malE gene, which encodes maltose-binding protein (MBP). This results in the expression of an MBP-fusion protein (1,2,3). The technique uses the strong Ptac promoter and the translation initiation signals of MBP to express large amounts of the fusion protein. The fusion protein is then purified by a one-step affinity purification specific for MBP (Figure 1) (4).

    The system uses the pMAL vectors which are designed so that insertion of a target gene results in an MBP fusion protein. pMAL-c5 series has an exact deletion of the malE signal sequence, resulting in cytoplasmic expression of the fusion protein. pMAL-p5 series contains the normal malE signal sequence, which directs the fusion protein through the cytoplasmic membrane. pMAL-p5 fusion proteins capable of being exported can be purified from the periplasm. Between the malE sequence and the polylinker there is a spacer sequence coding for 10 asparagine residues. This spacer insulates MBP from the protein of interest, increasing the chances that a particular fusion will bind tightly to the amylose resin. The vectors also include a sequence coding for the recognition site of a specific protease. This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-derived residues to the protein (5). For this purpose, the polylinker includes a restriction site superimposed on the sequence coding for the site of the specific protease, where the gene of interest is inserted. A number of other useful sites are present directly downstream to facilitate cloning.



    Figure 1: Schematic illustration of purification using pMAL system.
    Figure 2: 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.

    System Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pMAL-c5X Vector-20200 μg/ml
    pMAL-p5X Vector-20200 μg/ml
    Factor Xa Protease-201 mg/ml
    E. coli ER2523 (NEB Express) (Glycerol Stock)
    Amylose Resin4
    MBP5 Protein-2040 μg/ml
    MBP5-paramyosin-ΔSal-205 mg/ml
    Anti-MBP Monoclonal Antibody-201 mg/ml

    References

    1. Guan, C. et al. (1987). Gene. 67, 21-30.
    2. Maina, C.V. et al. (1988). Gene. 74, 365-373.
    3. Riggs, P.D. (1990). In F.M. Ausebel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K.Struhl (Ed.), Current Protocols in Molecular Biology. 16.6.1-16.6.10. New York: John Wiley & Sons, Inc.
    4. Kellerman, O.K. and Ferenci, T. (1982). In W.A. Wood(Ed.), Methods in Enzymology. 90, 459-463. New York: Academic Press.
    5. LaVallie, E.R. and McCoy, J.M. (1990). In F.M. Ausebel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K.Struhl (Ed.), Current Protocols in Molecular Biology. 16.4.1-16.4.17. New York: John Wiley & Sons, Inc.
    6. Kapust and Waugh (1999). Protein Science. 8, 1668-1674.

    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. Where can I find many more detailed FAQs for pMAL Protein Fusion & Purification System (E8200)?
    2. What strain(s) do you recommend as hosts for the pMAL vectors?
    3. What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector?
    4. What are some of the possible explanations for an inability to clone an insert into a pMAL vector?
    5. When we analyze our fusion protein expression by Western blot using the Anti-MBP Monoclonal Antibody, only a small fraction of the protein is full-length, while most of it migrates close to the MBP5* marker.
    6. My fusion protein is insoluble; is there anything I can do to get it expressed as soluble protein?
    7. Much of my fusion protein flows through the amylose column. Is there anything I can do to improve my fusion’s affinity for the amylose column?
    8. How many times can I use the amylose column?
    9. How should I store my protein after it is purified?
    10. What is MBP5? Is it different from wild-type MBP produced from E. coli?
    1. Cloning a PCR Fragment Into a pMAL Expression Vector (E8200)
    2. Purification of a Fusion Protein generated by The pMAL Protein Fusion and Purification System (E8200)
    3. Cleavage of the Fusion Protein Generated Using The pMAL Protein Fusion and Purification System (E8200)
    4. Separating the Protein of Interest from MBP after Protease Cleavage Using The pMAL Protein Fusion and Purification System (E8200)
    5. Quick Start Guide (E8200)

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