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.
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.
Part of The Investigator's Annexe program.
|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.|
Source: Wiley Video Abstract, Biotechnology and Bioengineering DOI: 10.1002/bit.24605
Purity and Demonstrated mSA Function
(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).
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