Engineered Monomeric Streptavidin 2 (mSA2) Protein

Monomeric Streptavidin 2 (mSA2) is an engineered monomeric streptavidin protein that binds biotin with Kd ~ 2 – 3 nM. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only.

Highlights:

  • Monomeric form allows for association with biotinylated ligands without inducing aggregation through multivalent interaction
  • Smaller MW compared to wild type streptavidin is advantageous in applications where access to biotinylated ligands may be a concern
  • Moderate affinity for biotin compared to wild-type strepavidin useful for subsequent dissociation of the bound ligand
  • Can be used as a genetic fusion tag, in which mSA2 can be fused to cell surface receptors and labeled with biotinylated fluorophores

Streptavidin is widely used in biotechnology and molecular research for detection, purification, crosslinking, and labeling of biotinylated targets. However, wild-type streptavidin is an obligate tetramer and can crosslink biotinylated targets. Target aggregation is a significant obstacle in some applications, including the labeling of biotinylated cell surface receptors, where crosslinking can perturb protein stability and function.

From the laboratory of Sheldon Park, PhD, University at Buffalo.

The Investigator's Annexe Part of The Investigator's Annexe program.

Catalog Number Product Size AVAILABILITY Price Qty
EBU001
Engineered Monomeric Streptavidin 2 (mSA2) Protein, 1mg
1mg Currently Unavailable
Price: $213.00
Contact us for details.
EBU002
Engineered Monomeric Streptavidin 2 (mSA2) Protein, 3mg
3mg Currently Unavailable
Price: $450.00
Contact us for details.
Specifications
Product Type: Protein
Name: Monomeric Streptavidin (mSA) 2
Source: Recombinant, purified from E. coli
Molecular Weight: 15.6 kDa (including FLAG tag)
Extinction Coefficient: E280= 37530 M-1cm-1
Amino Acid Sequence: TSHHHHHHEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSDYKDDDDK
Fusion Tag(s): N-terminal 6xHis tag, C-terminal FLAG (DYKDDDDK) tag
Purity: >90% by SDS-PAGE
Buffer: 50 mM Tris (pH 7.5), 150 mM NaCl
Solubility: >30mg/mL in PBS
Storage: Store at 4C up to 1 ? 2 months. Flash freeze in liquid nitrogen and place at -80C for long-term storage. Avoid repeated freeze-thaw cycles.
Shipped: Cold packs
Research

Source: Wiley Video Abstract, Biotechnology and Bioengineering DOI: 10.1002/bit.24605

Data

Purity and Demonstrated mSA Function

mSA2 Figrure

(A) SDS-PAGE analysis of purified mSA2 protein. (B) HEK293T cells were transfected with mSA-EGFP-TM or with SA-EGFP-TM to display either monomeric or wt streptavidin on the cell surface. The cells were then incubated with b-PE to test if the displayed streptavidin can bind biotin. Although both (i) mSA and (ii) SA constructs result in EGFP expression, only mSA-displaying cells are able to bind b-PE. The labeling is specific because untransfected cells (iii) are neither GFP nor PE positive. The SA EGFP image was collected with a 40% higher gain compared to that of mSA, because the fluorescence was not detectable otherwise. DAPI staining was used to locate the nucleus.

Adapted from: Lim, K.H., et al. Biotechnology Bioeng 110, 57-67 (2013).

Provider
From the laboratory of Sheldon Park, PhD, University at Buffalo.
Comments
Estimated pI ~6.11. Sufficient for binding ~17 µg biotin per mg mSA2. Avoid high concentration of a reducing reagent (e.g. DTT >1 mM). mSA2 protein is present in 50 mM Tris buffer. Buffer exchange to another buffer (e.g. PBS) may be required for use with dye conjugation studies where Tris buffers are known to interfere.
References
  1. Lim, K.H., Huang, H., Pralle, A., and Park, S. Stable, High-Affinity Streptavidin Monomer for Protein Labeling and Monovalent Biotin Detection. Biotechnology Bioeng 110, 57-67 (2013)
  2. DeMonte, D., Drake, E., Lim, K.H., Gulick, A., Park, S., Structure based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins: Structure, Function, and Bioinformatics (DOI: 10.1002/prot.24320)
  3. Phelps KJ, Ibarra-Soza JM, Tran K, Fisher AJ, Beal PA. Click modification of RNA at adenosine: structure and reactivity of 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenosine in RNA. ACS Chem Biol. 2014 Aug 15;9(8):1780-7. View Article
  4. Castellanos MM, Snyder JA, Lee M, Chakravarthy S, Clark NJ, McAuley A, Curtis JE. Characterization of Monoclonal Antibody-Protein Antigen Complexes Using Small-Angle Scattering and Molecular Modeling. Antibodies (Basel). 2017;6(4). pii: 25. View Article

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