How Does Streptavidin Work? Function, Structure, & Uses

Two links in a chain connected to each other

Streptavidin: Overview

Streptavidin is a tetrameric protein isolated from the bacterium Streptomyces avidinii. It’s highly known for its lesser non-specific binding with the biotin (or Vitamin H) molecule. The protein has a molecular weight of 60 kDa. 

Streptavidin tetramer structure is composed of four monomeric subunits, with each having a high affinity for biotin with a dissociation constant of ~10−14 mol/l. Its homologs have been identified in a range of organisms, including bacteria, fungus, chickens, and frogs. 

The binding of biotin to streptavidin is considered one of the strongest non-covalent bondings. This binding affinity is around 103-106 times higher than the antigen-antibody interactions. Avidin is another protein having high binding affinity and specificity for biotin. However, its affinity is comparatively lower than that of streptavidin, mainly because avidin is glycosylated and has a carbohydrate-binding site in its molecular structure.

Figure: Monomeric streptavidin bound with biotin.

The streptavidin-biotin complex has high thermal stability. It also possesses strong resistance to denaturants, organic solvents, proteolytic enzymes, detergents, and extreme pH and temperature. These characteristics serve as the foundation for its wide application in different industries.

The streptavidin-biotin interaction is extensively exploited in biotechnology, biochemistry, and molecular biology labs and industries. It serves as an immunological tool in a range of assays, such as enzyme-linked immunosorbent assays (ELISA), western blotting, and immunohistochemistry. 

In this article, we will review the working mechanism of streptavidin, learn about its conjugates, and its applications in Life Sciences labs in a spectrum of lab workflows.

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Streptavidin vs Avidin

Streptavidin and avidin, both proteins have a binding affinity for the biotin vitamin. However, the affinity strength differs based on some structural differences. The problems with ABC (avidin-biotin complex) system challenges can be overcome by the B-SA (biotin-streptavidin) method because of many characteristic differences between both the complexes:

  • As streptavidin contains no carbohydrates, it cannot bind to lectin-like substances (non-specific binding) in normal tissue, including the kidney, liver, brain, and mast cells.
  • Streptavidin conjugates exhibit less nonspecific electrostatic binding than avidin conjugates because their isoelectric point is close to neutrality — avidin’s isoelectric point is 10. The conjugates can be HRP (horseradish peroxidase), luciferin phosphate, and AP (alkaline phosphatase) substrates in both cases.
  • B-SA system is a highly stable reagent due to the enzyme’s direct conjugation to streptavidin, which allows for dilution and storage for a long time as a ready-to-use product (RTU). 

This principle can be applied to secondary and labeling reagents to increase sensitivity and also allow greater dilution of expensive primary antibodies. The enzyme label may either be peroxidase or alkaline phosphatase.

Streptavidin Structure and Function

Streptavidin is a tetrameric structure, composed of four monomer units. Each monomer subunit consists of eight twisted antithetical beta strands, forming tertiary barrel structures. The residues present at the interior side of the barrel form a biotin-binding site with a conserved Trp120 neighboring subunit. Thus, every subunit interacts with its adjacent subunit to form its biotin-binding site.

The protein consists of 159 amino acids, of which the first 24 amino acids serve as the signal peptide. During the binding of the protein, the ends are cleaved to access the core for increased biotin-binding affinity. 

The streptavidin used in labs is a recombinant monomeric streptavidin form, which is produced and expressed in Escherichia coli (E. coli). The expression system used for protein production includes yeast, bacterial, plant, and mammalian cells.

Figure: Tetrameric structure of streptavidin bound with biotin vitamin.

What is Streptavidin Used For?

Streptavidin-biotin complex has extensive applications in biochemistry and biotechnology labs in a spectrum of workflows, such as ELISA, Western Blotting, and other Immunoassays. A few examples of the functional roles played by the molecule in the workflows are as follows:

  • This molecule serves as a linker between molecules like monoclonal antibodies and radioisotopes. These molecules have been used in disease diagnosis and treatment, such as cancer.  
  • Monovalent streptavidin is used in fluorescent tracking of decorating DNA origami, cell surface receptors, and identifying specific regions for cryo-electron microscopy by acting as a pointer.

Not only in combination with biotin but streptavidin alone have many other applications as a generic receptor to bind engineered ligands. For example, streptavidin-binding DNA and RNA aptamers have been identified in nucleic acid libraries for use in purification or fluorescent detection. 

Protein Purification and Detection

Purification and detection of natural and recombinant proteins and various other biomolecules are one of the most common applications of streptavidin. It’s done by using the interaction to attach these molecules to one another or the solid surface.

As the biotin and streptavidin interaction is strong, it requires harsh conditions to break that bond. It also results in the denaturation of the protein. However, short incubation in water above 70°C reversibly breaks the interaction (at least in the case of biotinylated DNA) without denaturing streptavidin, allowing it to be reused.

Immunoassays

A wide variety of immunohistochemistry, fluorescence imaging techniques, and multicolor flow cytometry use fluorescent streptavidin conjugates to detect biotinylated ligands, antibodies, and DNA probes.

Figure: An illustrative diagram of the B-SA system in antibody detection.

What Industries Use Streptavidin? 

Streptavidin is used in a variety of Life Sciences labs and industries to perform a myriad of lab workflows. Some of the examples are given below:

  • The enzymatic biotinylation has application in the studies of protein–RNA, protein-protein, PTM, and protein–DNA interactions. 
  • Fluorescence in situ hybridization (FISH), microarrays, DNA arrays, and other hybridization techniques utilize biotin-modified nucleoside triphosphate analogs, such as dCTP and dUTP.
  • The labeling techniques that utilize enzymatic non-radioactive DNA labeling reactions, involving streptavidin, include PCR, 3”-end labeling, nick translation, cDNA labeling, and random prime labeling. 

An 11-, 14-, 16- or 20-atom spacer between the molecules and biotin allows fluorophores and enzyme streptavidin conjugates to detect the protein and amplify the signal on agarose and magnetic beads.

Nanobiotechnology 

Streptavidin has a major application in nanobiotechnology. It has been used with biomolecules like lipids and proteins to develop nanoscale structures or devices. For example, streptavidin can be used to link a polymer of biotinylated nucleic acids (such as DNA) to form scaffolds for single-walled carbon nanotubes.

Furthermore, antigen detection is carried out using streptavidin beads immobilized with biotinylated antibodies. The beads are prepared by purification of the protein, chemical cross-linking, and protein production.

The engineered streptavidin polymer beads also have applications in enzyme immobilization, DNA purification, enzyme-linked immunosorbent assay (ELISA), and flow cytometry processes.

Figure: Application of streptavidin in drug delivery by conjugating to different types of molecules.

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Streptavidin is a tetrameric molecule having a high binding affinity for the biotin vitamin. The molecule is made of four monomers with each having a biotin-binding site. The streptavidin-biotin interaction has a wide range of applications in biotech and biochem labs. It’s exploited in lab workflows like ELISA, western blotting, and immunoassays.

Protein biotinylation kits, reactive-biotin derivatives, Colorimetric Biotin Quantitation Kit, and many such kits are commercially available for their use in lab workflows and industries. However, it’s advised to go through the product safety data sheet (SDS) to ensure the quality of the product and the results obtained using them.

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