Streptavidin Mutein Matrix SAVSBPM18

Streptavidin SBPM18 is a streptavidin mutein that can bind both biotinylated proteins and SBP tagged proteins with high affinity (Kd ~10-8M) in a reversible manner.

Highlights:

  • Provided as SBPM18 mutein-Sepharose CL-6B affinity matrix
  • Ideal for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags
  • Target proteins can be affinity purified in high purity and with excellent recovery
  • Mild conditions are used for elution of target proteins and regeneration of the column
  • Many buffers can be used for in the binding, washing and elution steps
  • The affinity matrix is reusable

Streptavidin binding peptide (SBP) tag is a peptide tag that binds to streptavidin with nmolar binding affinity (2). Although the original peptide tag is 38 amino acids in length, crystallographic and biochemical studies indicate that only 25 amino acids are essential for high affinity binding (3). It has high binding affinity to streptavidin because both the N- and C-terminal ends of the 25-amino-acid tag can bind simultaneously to the binding pockets in streptavidin, a tetrameric protein with a binding pocket in each subunit (3). One streptavidin can bind two SBP tags. Since the peptide binding pocket in each streptavidin subunit is the same as the biotin binding pocket in the subunit, biotin binding can effectively displace the bound SBP tag. Natural streptavidin captures biotin extremely tight (Kd ~10-14M). This streptavidin mutein has two mutations to weaken the interactions between biotin and streptavidin (1). This leads to biotin binding in a reversible manner. To simplify the preparation of the affinity matrix, the streptavidin mutein is fused to an agarose binding domain (4). The SAVSBPM18-ABD fusion binds tightly to Sepharose CL-6B. Affinity matrices prepared in this manner show comparable properties as matrices prepared via chemical coupling.

From the laboratory of Sui-Lam Wong, PhD, University of Calgary.

Catalog Number Product Size AVAILABILITY Price Qty
EUC012
Streptavidin Mutein Matrix SAVSBPM18, 1.5mL
1.5mL 1-2 weeks
Price: $199.00
Specifications
Name: Streptavidin M18-ABD Sepharose CL-6B
Format: Preswollen gel matrix in a 50% suspension. A two ml suspension can generate a column with 1 ml packed bed volume
Tested Applications: Affinity chromatography purification of biotinylated proteins and SBP (streptavidin binding tag) tagged proteins
Comments: Particle Size/Mesh: 45-145 um. Majority is around 50 um in diameter
Binding Capacity: 700 ug biotinylated BSA (12 biotin/BSA)/ml matrix
Resin Reconstitution: After packing the matrix to the column, rinse the column with TBS (50 mM Tris, 150 mM NaCl pH 7.2).
Recommended Mobile Phase: TBS (50 mM Tris, 150 mM NaCl pH 7.2)
Storage: 2-4 degree (C) in TBS
Shipped: Cold Packs
Research
Biotinylated proteins can be generated via enzymatic method (e.g. use the E. coli biotin ligase to biotinylate AviTag fusion proteins). This approach will generate biotinylated proteins with one biotin/protein. Chemical biotinylation usually results in multiple biotin groups per protein. Biotinylated proteins with multiple biotin groups per molecule will offer tighter binding. BSA with 12 biotin groups per protein can be effectively eluted off from this matrix under very mild elution conditions.
Provider
From the laboratory of Sui-Lam Wong, PhD, University of Calgary.
Comments

Very mild conditions (washing with a buffer containing biotin) are sufficient to elute the bound target proteins from the matrices. Extensive wash of the column with the buffer without biotin can effectively regenerate the streptavidin mutein matrix for the next round of purification.

See references 1 and 4 for detailed operating conditions.

References
  1. Wu, S. C. & Wong, S. L. Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins. PloS one 8, e69530, doi:10.1371/journal.pone.0069530 (2013).
  2. Keefe, A. D., Wilson, D. S., Seelig, B. & Szostak, J. W. One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr.Purif. 23, 440-446 (2001).
  3. Barrette-Ng, I. H., Wu, S. C., Tjia, W. M., Wong, S. L. & Ng, K. K. The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits. Acta crystallographica. Section D, Biological crystallography 69, 879-887, doi:10.1107/S0907444913002576 (2013).
  4. Wu, S. C., Wang, C., Hansen, D. & Wong, S. L. A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix. Sci Rep 7, 42849. doi: 10.1038/srep42849 (2017).

If you publish research with this product, please let us know so we can cite your paper.

 
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