His-Strep pQE-TriSystem Vector Set

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

  • 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
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.
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His-Strep pQE-TriSystem Vector Set
pQE-TriSystem His-Strep 1 and pQE-TriSystem His-Strep 2 vectors, 25 µg each
 32942
$766.00
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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.
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Highly sensitive detection of proteins carrying a Strep-tag.|Two-step affinity purification procedure.|Ultrapure protein in two steps.|
Dot blot showing the sensitivity of Strep-tag Antibody. The indicated amounts of protein were spotted onto a membrane and detected using Strep-tag Antibody, an anti-mouse secondary antibody conjugated to horseradish peroxidase, and the ECL chemiluminescent detection system. GFP: green fluorescent protein.||Thioredoxin, expressed in NIH-3T3 cells, was purified using the two-step affinity purification system. [A] Coomassie-stained gel. [B] Silver-stained gel.|
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").
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.

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").

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
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