Product Class: Other

pMAL-c6T Vector

This product is a replacement for NEB #N8108, pMAL™-c5X Vector


Catalog #N0378

Product Introduction

The pMAL-c6T Vector is designed to produce maltose-binding protein (MBP) fusions in the cytoplasm. The MBP has been engineered for tighter binding to amylose resin. The vector expresses an N-terminal his-tagged malE gene followed by a multiple cloning site containing a TEV protease recognition sequence, allowing MBP to be cleaved from the protein of interest after purification. 

  • View sequence details
  • pMAL-c6T is the recommended vector for use with the NEBExpress® MBP Fusion and Purification System (NEB #E8201)
  • Fusion to MBP significantly enhances the solubility and proper folding of proteins expressed in E. coli (1)
  • Vector includes His-tag and TEV Protease recognition site, allowing the protein of interest to be cleaved from MBP using TEV Protease (NEB #P8112)
  • Two-step purification: amylose resin elution followed by TEV Protease cleavage and Ni resin isolation results in a highly-pure, tag-free target protein
  • MBP has been engineered for tighter binding to amylose resin
  • Ampicillin resistant
 

Product Information

Description

The pMAL-c6T Vector provides a method for producing a protein expressed from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein in the cytoplasm (1,2). The MBP has been engineered for tighter binding to amylose resin. The method uses the strong “tac” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP’s affinity for maltose (5). The vector expresses an N-terminal hexahistidine tagged malE gene (lacking its secretory signal sequence) followed by a multiple cloning site containing a TEV protease recognition sequence and stop codons in all three frames. This allows MBP to be cleaved from the protein of interest by TEV Protease after purification. The vector also carries the lacIq gene, which encodes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG induction. 

Source: NEB-10 beta competent E. coli (pMAL-c6T)

Figure 1: pMAL-c6T Vector



The pMAL- c6T Vector has an exact deletion of the malE signal sequence. Arrows indicate the direction of transcription. Unique restriction sites in the multiple cloning site (MCS) are indicated.

Product Source

NEB-10 beta competent E. coli (pMAL-c6T)
This product is related to the following categories:
NEBExpress MBP Fusion and Purification System,
Bacterial E. coli Protein Expression Products,
DNA Plasmids & Substrates Products,
Protein Expression 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

Affinity Tag

Maltose-Binding Protein (MBP)

Sequence Files

Fasta GenBank

Features

  • The recommended vector for use with the NEBExpress® MBP Fusion and Purification System (NEB #E8201)
  • Fusion to MBP significantly enhances proper folding and solubility of target proteins
  • Vector includes His-tag and TEV Protease recognition site
  • Two-step purification: amylose resin elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure, tag-free target protein

Product Notes

  1. A detailed product manual for the NEBExpress MBP Fusion and Purification System with protocols can be found here.
  2. NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagation and subcloning. NEB Express Competent E. coli (High Efficiency) (NEB #C2523) is recommended for expression using this vector.
  3. This vector conveys ampicillin resistance for propagation in E. coli.

References

  1. Guan, C. et al (1987). Gene. 67, 21–30.
  2. Maina, C.V. et al (1988). Gene. 74, 365–373.
  3. Amann, E. et al (1985). Gene. 40, 183–190..
  4. Duplay, P. et al (1984). J. Biol. Chem. 259, 10606–10613.
  5. 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. NEBuilder Assembly of a PCR Fragment
  4. Transformation Protocol (NEB #E8201)
  5. Small Scale Affinity Chromatography (NEB #E8201)
  6. Large Scale Affinity Chromatography (NEB #E8201)
  7. Cleavage of the Fusion Protein
  8. Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)

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

Web Tools

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. What is the DNA sequence of this product?
  5. What are the differences between the generations of pMAL vectors (e.g. pMAL-c2 vs. pMAL-c5 vs. pMAL-c6 or pMAL-p2 vs. pMAL-p5)?

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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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