Fluorophore-friendly streptavidin

Oxford researchers have developed a new streptavidin mutant, Flavidin, which was designed using amine landscaping and provides precise positioning of amine-reactive dyes. Using Flavidin (fluorophore-friendly streptavidin), the ultra-tight biotin-binding properties are retained after dye modification, unlike parental streptavidin. This mutant also has enhanced brightness and specificity for cellular and biochemical applications.

Oxford researchers have developed a new streptavidin mutant, Flavidin, which was designed using amine landscaping and provides precise positioning of amine-reactive dyes. Using Flavidin (fluorophore-friendly streptavidin), the ultra-tight biotin-binding properties are retained after dye modification, unlike parental streptavidin. This mutant also has enhanced brightness and specificity for cellular and biochemical applications.

This new variant is entirely genetically encoded with no use of alternative amino acids and can be produced using the same methods as other recombinant streptavidin. It should have extensive use in biological research, diagnostics and biotechnology.

Biotin/Streptavidin:

  • One of the most widely used protein-ligand interactions in molecular biology and diagnostics
  • Exceptionally stable affinity of streptavidin for biotin is one of the strongest known non-covalent interactions
  • Resistant to organic solvents, denaturants, detergents, proteolytic enzymes and temperature/pH extremes
  • Extensive number of lab applications

Chemical modification of streptavidin through widely-used amine-reactive probes results in significant impairment of biotin binding properties, decreasing sensitivity. In addition, dye-labelling often leads to attachment of excess dye molecules, which can decrease fluorescence and promote non-specific binding to cellular components, reducing signal to noise. By removing all the lysines in streptavidin, the researchers have achieved more specific targeting of the amine-reactive dye, to produce increased brightness without affecting biotin-binding. Less dye modification also decreases damage to protein folding, as well as dye-induced non-specific binding, minimising noise.

Flavidin system applications:

Flow cytometry/ fluorescence microscopy – streptavidin linked to fluorescent dyes is used to detect biotinylated antibodies or nucleic acids. Brightness is crucial, particularly for poorly expressed targets. The Flavidin system will improve detection against the auto-fluorescent cellular background.

T cell analysis and purification – MHC I/II molecules are tetramerised by streptavidin. These tetramers are a central tool for immunology/vaccinology to monitor T cell responses. Dye conjugation currently produces poor results, however, the Flavidin system should allow monitoring of several different T cell specificities in a single sample, by using multiple dyes.

B cell analysis and purification – B cell antigens are often multimerised by streptavidin and used for the identification of B cell populations expressing antibodies against a target. This is useful for analysis of the immune response and to identify antibodies which may be useful for future therapies for indications such as HIV and malaria.

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