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.
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.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|E. coli ER2523 (NEB Express) (Glycerol Stock)||-20|
|Anti-MBP Monoclonal Antibody||-20|
|MBP5 Protein||-20||40 µg/ml|
|pMAL™-c5X Vector||-20||200 µg/ml|
|pMAL™-p5X Vector||-20||200 µg/ml|
|Factor Xa Protease||-20||1 mg/ml|
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