This is a manual for NEBExpress® MBP Fusion and Purification System.
The NEBExpress MBP Fusion and Purification System (NEB #E8201S) is designed for the expression and purification of proteins produced from cloned genes. The system utilizes the pMAL-c6T vector, which expresses a maltose-binding protein (MBP) fusion protein containing an N-terminal hexahistidine tag. This allows for high-level expression and one-step purification using amylose resin affinity chromatography.
The manual details the workflow from constructing the fusion plasmid to the final purification and cleavage of the protein of interest. Key components include the Amylose Resin Column, TEV Protease for cleaving the MBP tag, and various buffers. The document also covers subcloning strategies, PCR fragment preparation, transformation protocols, and troubleshooting tips.
| Binding capacity | > 4 mg/ml bed volume |
|---|---|
| TEV Protease activity | 1 unit cleaves 2 µg of MBP-fusion protein to 95% completion in 1 hour at 30°C |
| Anti-MBP Antibody dilution | 1:10,000 |
| MBP6 Protein Molecular Weight | 45,519 daltons |
| MBP6-TEV-Paramyosin DSal Molecular Weight | 70,103 daltons |
| Fusion protein yield range | 10–40 mg/liter |
| Maximum fusion protein yield | 200 mg/liter culture |
The suggested dilution for Western blotting or ELISA is 1:10,000.
The Amylose Resin should be stored at 4°C, while all other reagents should be stored at –20°C.
Cells can be grown at 37°C for 2 hours, 30°C for 4 hours, room temperature for 6–8 hours, or overnight at 12–16°C.