Streptavidin

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Streptavidin (SA) is a 56kDa homotetramer of Streptomyces avidinii, which can bind to four biotin molecules with K_d~10^-14 M. The protein also has high thermal stability (Tm for apo-SA: 73°C, Tm for biotin-SA: 112°C), and is resistant to many extreme conditions such as pH, denaturants and enzymatic degradation, etc. Streptavidin homologs have been identified from organisms such as fungus, bacteria, frogs and chickens. Since this molecule can tolerate mutations, it can serve as a good platform for engineering research. At present, a large number of streptavidin mutants have been reported in the literature, the mutations include single residue substitution that predictably changes the glycosylation pattern of chicken-derived avidin, the rational design of neo host-guest system for organometallic chemistry applications, and large-scale protein engineering research, etc. A large number of studies have proved the research interest of scientists on the streptavidin-biotin interaction and its applications. In short, the improvement of chemical and enzymatic biotinylation technology and the newly engineered streptavidin variants have played important roles in scientific progress and technological development.

Fig 1. Structure of monovalent streptavidin (Zhang, M.; et al. 2016)

Streptavidin-based enzymes

Streptavidin is a powerful platform for constructing novel enzymes because it has a robust structure and can tolerate extensive amino acid substitutions. The engineered hydrolase activity can be used to cut biotin affinity purification tags. The binding of biotin is particularly useful for engineering enzyme capabilities because it can be used to recruit and maintain reactive components in the desired spatial orientation. By anchoring biotinylated inorganic cofactors in the binding pocket, various metalloenzymes have been successfully engineered on streptavidin scaffolds. Studies have reported a hybrid streptavidin catalyst containing a fixed metal center, indicating that powerful protein engineering techniques such as directed evolution can be used to evolve streptavidin-based enzymes. The engineered streptavidin enzymes can be used to create cellular pathways to increase the microbial production of high-value chemicals used in pharmaceutical and industrial applications. The ability to use artificial and natural catalysts to introduce synthetic reaction cascades into model organisms demonstrates the potential application of streptavidin-based metalloenzymes in metabolic engineering.

Pretargeted cancer therapy

The interaction between streptavidin and biotin can be used in drug delivery systems for cancer and gene therapy. It can effectively increase the therapeutic index of a treatment because of its ability to separate the targeting agent from the actual drug. For example, in cancer therapy, biotinylated antibodies against known cancer biomarkers are used to recruit radiolabeled streptavidin. By removing unbound antibodies from the patient before adding streptavidin, the contrast of radiation exposure between cancer and normal tissue can be further optimized. A series of variants of this method have been developed. In addition to radionuclides, the cargo delivered to tumors can also include liposomes containing chemotherapeutic agents. However, the challenge of streptavidin immunogenicity still exists in the drug targeting system based on streptavidin. At present, there have been studies on mutations that reduce the immunogenicity of streptavidin.

Fig 2. Streptavidin-based strategies for targeted drug delivery (Dundas, C.M.; et al. 2013)

Streptavidin-based sensors

Streptavidin is a powerful tool for the development of novel sensors due to its high biotin affinity. Recent studies have reported trisbiotinylated oligonucleotides (so-called “spiders”) that can simultaneously bind to the three binding sites of streptavidin. Because the designed molecule can be used to construct monovalent streptavidin from wt tetramer, this work has received a lot of attention. Its binding site retains high biotin affinity, so the complex can be used in various applications that require high-affinity monovalent biotin binding. Interestingly, it was shown that the addition of single-stranded DNA complementary to the spider sequence induces the dissociation of one of the bound biotins, which can be detected d by ELISA using horseradish peroxidase-conjugated streptavidin. A single mismatch reduces the rate of biotin dissociation, indicating that the complex can be used as a sensor for detecting specific sequence oligonucleotides. At present, some other sensors that do not need to be combined with biotin are also reported, they use streptavidin as their generic receptor to bind to engineered ligands.