This product is a direct replacement for NEB #E8200, pMAL™ Protein Fusion and Purification System
Catalog #E8201
The NEBExpress® MBP Fusion and Purification System takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein in E. coli. The resulting product is an MBP fusion protein, which is then purified by affinity chromatography.
Reliable E. coli expression: substantial yields (up to 100 mg/L)
Fusion to MBP has been shown to enhance the solubility of proteins expressed in E. coli (1)
Two-step purification: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target protein
Gentle elution with maltose; no detergents or harsh denaturants required
Product Information
In the NEBExpress® MBP Fusion and Purification System, the pMAL-c6T vector provides a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from and in frame with the malE gene of E. coli, which encodes maltose-binding protein (MBP); this construct results in the expression of an MBP fusion protein (2,3). The pMAL-c6T vector expresses the N-terminal hexahistidine tagged malE gene (lacking its secretory signal sequence and engineered for tighter binding to amylose) followed by a multiple cloning site containing a TEV protease recognition sequence and stop codons in all three frames. The pMAL-c6T vector expresses the MBP fusion in the cytoplasm. The method uses the strong “tac” promoter and the malE translation initiation signals to yield high-level expression of the cloned sequences (4,5). The fusion protein is then purified by a one-step purification method using amylose resin and MBP’s affinity for maltose(6).
Following amylose purification, the target protein can be cleaved from the MBP-tag using TEV Protease, without adding any vector-derived residues to the protein. Both the MBP-tag and TEV Protease are polyhistidine-tagged for easy removal from the reaction. Loading the digest onto NEBExpress Ni Resin (NEB #S1428) sequesters both the MBP-tag and TEV Protease, thereby isolating the target protein in the column flow through. The target protein yield can be up to 100 mg/L, with typical yields in the range of 10–40 mg/L.
Figure 1: Schematic illustration of the NEBExpress MBP Fusion and Purification System
Figure 2. Protein Expression using the NEBExpress MBP Fusion and Purification System
SDS-polyacrylamide gel electrophoresis of fractions from the affinity purification of MBP6-TEV-Paramyosin ΔSal. Lane 1: Protein Standard. Lane 2: uninduced cells. Lane 3: induced cells. Lane 4: purified fusion protein eluted from amylose column with maltose. Lane 5: purified protein after TEV Protease cleavage. Lane 6: target protein isolated from NEBExpress Ni Resin flow through.
This product is related to the following categories:
A detailed product manual, with protocols can be found here.
Recommended long term storage (>30 days) for E. coli ER2523 (NEBExpress) is -80°C.
The amylose resin should be stored at 4°C to prevent damage from freezing. However, the performance of the resin is not degraded by one freeze/thaw cycle.
MBP6-TEV-Paramyosin-ΔSal is a positive control for TEV Protease cleavage.
References
Kapust and Waugh (1999). Protein Science. 8, 1668-1674.
Guan, C. et al. (1987). Gene. 67, 21–30.
Maina, C.V. et al. (1988). Gene. 74, 365–373.
Amann, E. et al. (1985). Gene. 40, 183–190.
Duplay, P. et al. (1984). Biol. Chem.. 259, 10606–10613.
Kellerman, O.K. et al. (1982). Methods in Enzymology. 90, 459–463.
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.
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