Product Class: Kit

NEBExpress® MBP Fusion and Purification System

This product is a direct replacement for NEB #E8200, pMAL™ Protein Fusion and Purification System


Catalog #E8201

Product Introduction

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
 
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Product Information

Description

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:
Amylose Purification (MBP-tag),
NEBExpress MBP Fusion and Purification System,
Non-T7 Expression,
Bacterial E. coli Protein Expression Products,
Affinity Purification Products,
Protein Purification Products,
Protein Expression Products,
E. coli Expression Strains Products,
This product can be used in the following applications:
Non-T7 Expression,
Target Protein Insolubility ,
Expression of Difficult Proteins,
Protein Expression in E. Coli,
Protein Expression

Properties & Usage

Features

  • Reliable E. coli expression: substantial yields (up to 100 mg/L)
  • Fusion to MBP significantly enhances proper folding of target proteins
  • 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 Notes

  1. A detailed product manual, with protocols can be found here.
  2. Recommended long term storage (>30 days) for E. coli ER2523 (NEBExpress) is -80°C.
  3. 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.
  4. MBP6-TEV-Paramyosin-ΔSal is a positive control for TEV Protease cleavage.

References

  1. Kapust and Waugh (1999). Protein Science. 8, 1668-1674.
  2. Guan, C. et al. (1987). Gene. 67, 21–30.
  3. Maina, C.V. et al. (1988). Gene. 74, 365–373.
  4. Amann, E. et al. (1985). Gene. 40, 183–190.
  5. Duplay, P. et al. (1984).  Biol. Chem.. 259, 10606–10613.
  6. Kellerman, O.K. et al. (1982). Methods in Enzymology. 90, 459–463.

Protocols, Manuals & Usage

Protocols

  1. NEBExpress MBP Fusion and Purification System Quick Start Protocol (NEB #E8201)
  2. Cloning a PCR Fragment Into a pMAL Expression Vector (E8201)
  3. Transformation Protocol (NEB #E8201)
  4. Small Scale Affinity Chromatography (NEB #E8201)
  5. Large Scale Affinity Chromatography (NEB #E8201)
  6. Cleavage of the Fusion Protein
  7. Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)
  8. NEBuilder Assembly of a PCR Fragment

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What strain(s) do you recommend as hosts for the pMAL vectors?
  2. What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector?
  3. What is the minimum size of a fragment that can be cloned into pMAL and expressed fused to MBP? Can short peptide sequences (~ 10 amino acids) be added onto MBP?
  4. How many times can I use the amylose column?
  5. What is known about binding in the presence of nonionic detergents?
  6. Can I substitute a different buffer and/or salt concentration in the Column Buffer?
  7. Can I perform a batch purification using the amylose resin?
  8. Can MBP fusions be purified in the presence of denaturants like urea or guanidine-HCl?
  9. How can TEV Protease be removed from the reaction after cleavage?
  10. Is the rate of TEV Protease cleavage affected by urea, guanidine hydrochloride and/or SDS?
  11. How do I separate MBP and TEV Protease from the protein of interest?
  12. I want to rebind MBP to the amylose column, but the maltose must be removed. Can this be done by dialysis?
  13. How should I store my protein after it is purified?
  14. What is MBP6? Is it different from wild-type MBP produced from E. coli?
  15. What gravity column do you recommend?
  16. Is the amylose resin damaged by storage at -20°C?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

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.

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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