Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

Abstract

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.

Quantum dots–DNA bioconjugates: synthesis to applications

“…In order to further control the stoichiometry of biotin on streptavidin, molecularly engineered variants of the protein have been used, which have affinity for only one biotin [74,75] (and hence biotinylated DNA) that permit monolabelling ( per streptavidin molecule) with biotinylated rsfs.royalsocietypublishing.org Interface Focus 6: 20160064 DNA [76]. Molecular cloning has also realized facile production of higher affinity avidin variants such as dimeric rhizavidin [77,78] or electrostatically neutral variants (at physiological pH) such as neutravidin which further expands the tool-kit of this type of conjugation. Streptavidin-Bt -DNA conjugates of QD have been easily generated and could be used for various applications.…”

Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

“…To construct pCS2+-Cas9-mSA, the mSA coding sequence was amplified from pDisplay-mSA-EGFP-TM (a gift from Dr. Sheldon Park (Addgene plasmid #39863)) 3 and fused to the C-terminus of the Cas9 coding sequence with an optimized linker 4 in pCS2+-Cas9 using InFusion cloning. Supplementary Fig.…”

“…CRISPR/Cas9-mediated genome editing has shown consistently high efficiency in generating indels, point mutations and small insertions in zygotes 2,3 , largely making use of the non-homologous end-joining (NHEJ) and homology directed repair (HDR) pathways. However, the efficiency of precise introduction of large fragments by homologous recombination (HR) is still not ideal 4,5 .…”

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins

“…If two biotin moieties are appropriately positioned, one molecule of streptavidin could bind both of them, possibly complicating quantification. This could potentially be overcome by the use of monovalent forms of streptavidin (45). Furthermore, it would be necessary to use an excessive amount of streptavidin conjugate if an application would require unequivocal labeling of every click-reacted alkyne lipid molecule with the conjugate.…”