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 SystemFigure 2. Protein Expression using the NEBExpress MBP Fusion and Purification SystemSDS-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:
Amylose Purification (MBP-tag),
NEBExpress MBP Fusion and Purification System,
Non-T7 Expression,
Bacterial E. coli Protein Expression,
Affinity Purification,
Protein Purification,
E. coli Expression Strains Products,
Protein Expression
This product can be used in the following applications:
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