May 18, 2020
NEBExpress MBP Fusion and Purification System (NEB #E8201)
- 1New England Biolabs
External link: https://www.neb.com/protocols/2020/02/05/nebexpress-mbp-fusion-and-purification-system-quick-start-protocol-neb-e8201
Protocol Citation: New England Biolabs 2020. NEBExpress MBP Fusion and Purification System (NEB #E8201). protocols.io https://dx.doi.org/10.17504/protocols.io.bfayjifw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 19, 2020
Last Modified: May 18, 2020
Protocol Integer ID: 35896
Keywords: MBP, fusion, purification, E8201,
Abstract
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 inE. coli. The resulting product is an MBP fusion protein, which is then purified by affinity chromatography.
- ReliableE. coli expression: substantial yields (up to 100 mg/L)
- Fusion to MBP has been shown to enhance the solubility of proteins expressed inE. 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
Guidelines
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 themalEgene ofE. 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 taggedmalEgene (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 themalEtranslation 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.
Materials
MATERIALS
NEBExpress MBP Fusion and Purification SystemNew England BiolabsCatalog #E8201
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Subclone the gene of interest into the pMAL-c6T vector.
Grow cells containing the pMAL-c6T fusion plasmid in LB containing ampicillin and 0.2% glucose to an A600 of ~0.5.
Induce by adding IPTG to a final concentration of 0.3 millimolar (mM) .
Grow for an additional:
02:00:00 at 37 °C OR
04:00:00 at 30 °C OR
06:00:00 – 08:00:00 at Room temperature OR
Overnight at 12 °C to 16 °C .
Harvest the cells and either freeze immediately or resuspend in 25 ml column buffer (CB) per liter of culture.
Lyse the cells by freeze-thaw followed by sonication.
Clarify the lysate by centrifugation at 20000 rpm, 00:20:00 .
Dilute the supernatant (crude extract) by adding 125 mL cold CB for every 25 ml crude extract.
Load the diluted crude extract on a 15 ml amylose column.
Wash the column with ≥ 12 column volumes of CB.
Elute the fusion protein with CB containing10 millimolar (mM) maltose .