His-Strep pQE-TriSystem Vector Set

For parallel expression of His-Strep-tagged proteins in E. coli, insect, and mammalian cells

Features

  • Parallel expression in three systems with a single construct
  • Avoids the need for time-consuming subcloning
  • High-level expression
  • Tightly regulated expression
  • Enhanced stability of cytotoxic constructs
His-Strep pQE-TriSystem Vector Set

Cat. No. / ID: 32942

pQE-TriSystem His-Strep 1 and pQE-TriSystem His-Strep 2 vectors, 25 µg each
€504.00
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The His-Strep pQE-TriSystem Vector Set is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

Vectors in the His-Strep pQE-TriSystem Vector Set contain promoters for E. coli, insect-, and mammalian-cell expression systems. Proteins expressed from His-Strep pQE-TriSystem vectors carry a 6xHis- and a Strep-tag, enabling sequential purification on Ni-NTA and Strep-Tactin matrices to give ultrapure (>98% pure) protein. Both vectors encode a C-terminal tag that ensures only full-length protein is purified.

Performance

Double-tagged proteins expressed with the His-Strep pQE-TriSystem Vector Set can be isolated by two-step affinity purification to deliver ultrapure (>98% pure) protein (see figure " Ultrapure protein in two steps").
See figures

Principle

The His-Strep pQE-TriSystem Vector Set allows expression of proteins with both a 6xHis tag and the Strep-tag II. The double tag allows purification by a two-step affinity purification system, enabling simple and highly efficient purification of ultrapure proteins in a standardized procedure. The system also increases throughput by eliminating the need for development and optimization of protein-specific protocols. Recombinant proteins carrying both tags are purified sequentially on Ni-NTA and Strep-Tactin matrices (see figure " Two-step affinity procedure"). The two simple affinity purifications provide fully active, full-length, and ultrapure protein that is suitable for any downstream application.

See figures

Procedure

Recombinant proteins that carry two small affinity tags (the 6xHis tag and Strep-tag II) are efficiently expressed in E. coli, insect, or mammalian cells using pQE-TriSystem His-Strep vectors. After cell lysis and clearing of the lysate, proteins are initially purified using an immobilized-metal affinity chromatography (IMAC) procedure that is based on the proven 6xHis-tag-Ni-NTA interaction. After elution from the Ni-NTA matrix using imidazole, recombinant proteins (which also carry the Strep-tag II epitope) are loaded directly onto a Strep-Tactin matrix (see figure " Two-step affinity purification procedure"). No buffer exchange is required. Protein is eluted from the Strep-Tactin matrix using either biotin or desthiobiotin. Proteins can be detected with high specificity and sensitivity using mouse monoclonal Strep-tag or Anti·His antibodies (see figure " Highly sensitive detection of proteins carrying a Strep-tag").

See figures

Applications

The Two-Step Affinity Purification System is highly suited for applications where high purity is essential and is difficult to achieve with His-tag alone, for example, for proteins expressed in eucaryotic cells. The ultrahigh purity and convenience provided by the Two-Step Affinity Purification System make it the method of choice for:

  • Highly pure protein purification
  • Structural and functional analyses
  • Expression in eukaryotic systems

Supporting data and figures

Specifications

FeaturesSpecifications
expressionspeciesE. coli, mammalian, cells, insect cells
tagremovalsequenceNo
norcterminaltagN- and C-terminal tag
inframecloningnecessaryYes
tag6xHis tag
expressionIn vivo
allthreereadingframesprovidedNo

Resources

Selection Guides (1)
Vector Sequences & Maps (2)
For the pQE-TriSystem His-Strep 2 vector
For the pQE-TriSystem His-Strep 1 vector

FAQ

What is the basic technology behind the Strep-tag Protein Purification System?

The basic technology behind the Strep-tag System is a Two-Step Affinity Protein Purification using sequential purification of recombinant proteins carrying two affinity tags. Proteins that carry these two small tags (the 6xHis tag and the 8 amino acid containing Strep-tag) are efficiently expressed in E. coli, insect, or mammalian cells using pQE-TriSystem His·Strep vectors. After cell lysis and clearing of the lysate, proteins are initially purified by a procedure based on the proven 6xHis-tag-Ni-NTA interaction. After elution from the Ni-NTA matrix using imidazole, recombinant proteins (which also carry the Strep-tag epitope) are loaded directly onto a Strep-Tactin matrix (Strep-Tactin SuperFlow or Strep-Tactin Magnetic Beads). Protein is eluted from the Strep-Tactin matrix using either biotin or desthiobiotin. This two-step affinity purification delivers ultrapure (>98% pure) protein. The order of purifications can also be reversed (i.e., Strep-Tactin followed by Ni-NTA purification). Since purification is done under native conditions, the Two-Step Affinity Purification System can be beneficial for highly demanding applications (i.e., crystallization studies).

 

FAQ ID -740