The pMAL™ Protein Fusion and Purification System requires a cloned gene be 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. 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.
Expression in either the cytoplasm or periplasm: periplasmic expression can enhance folding of proteins with disulfide bonds
Fusion to MBP has been shown to enhance the solubility of proteins expressed in E. coli
Gentle elution with maltose; no detergents or harsh denaturants required
Reliable expression and substantial yields (up to 100 mg/L)
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-c5X has an exact deletion of the malE signal sequence, resulting in cytoplasmic expression of the fusion protein. pMAL-p5X 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 Factor Xa recognition site. 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 an XmnI site superimposed on the sequence coding for the Factor Xa cleavage site, 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 systemFigure 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:
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.
Kellerman, O.K. and Ferenci, T. (1982). In W.A. Wood(Ed.), Methods in Enzymology. 90, 459-463. New York: Academic Press.
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.
Kapust and Waugh (1999). Protein Science. 8, 1668-1674.
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].)
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Research Use Only
The buyer/user has a non-exclusive license to use the vector for Research Purposes Only. Commercial use of this vector requires a license from New England Biolabs, Inc.
U.S. Patent No. 5,643,758
Appln. No. WO2007/120809
Trademarks
NEW ENGLAND BIOLABS® and Q5® are registered trademarks of New England Biolabs, Inc. CUTSMART® and PMAL™ are trademarks of New England Biolabs, Inc.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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manualE8200
