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.
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Trademarks
NEW ENGLAND BIOLABS® and Q5® are registered trademarks of New England Biolabs, Inc. CUTSMART® and PMAL™ are trademarks of New England Biolabs, Inc.
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