Streptavidin

Streptavidin is a biotin-binding protein found in the culture broth of the bacterium Streptomyces avidinii. Like its namesake avidin, streptavidin binds 4 moles of biotin per mole of protein with a high affinity virtually unmatched in nature (Kd~10-15). Streptavidin lacks carbohydrate side chains present on avidin and has an isoelectric point nearer to neutrality where most useful biological interactions occur (pI of 5-6 vs 10 for avidin). As a result, streptavidin frequently exhibits lower levels of non-specific binding than does avidin when the proteins are used in applications relying upon the formation of avidin/biotin complexes (Chaiet and Wolf, 1964). ,   , In ELISA-based diagnostic systems, antibodies directed against a particular antigen may be covalently attached to reporter enzymes. Antigens are then quantitated by enzymatic assay after, binding to these conjugated molecules. The precise conditions for accomplishing such covalent attachments must be determined individually for each antibody/reporter combination, and often result in significant loss of either the enzymatic activity of the reporter enzyme or the binding functions of the antibodies. ,   , Streptavidin finds utility in these systems because antibody molecules are easily modified by the covalent attachment of derivatives of biotin with little or no loss in the ability of the antibody molecules to bind their antigens. These biotinylated antibodies may be detected by their interaction with conjugates of streptavidin and the reporter enzymes (Guesdon et al, 1979). The same preparation of conjugated streptavidin-reporter enzyme may be used with any number of different biotinylated antibodies making this system a highly flexible one. The reporter molecule may be bound to streptavidin covalently, or biotinylated and attached to streptavidin via the streptavidin-biotin interaction. Since streptavidin is multivalent (binding 4 molecules of biotin per tetrameric protein molecule) it may be used in combination with biotinylated antibody and biotinylated reporter enzymes to obtain amplified signals. Such amplification in ELISA's is otherwise difficult to obtain and requires the introduction of additional antibody components. ELISA systems employing streptavidin can readily detect nanogram, amounts of antigens. Composition: Streptavidin is a homotetramer. The protein contains no cysteine residues, carbohydrate side chains or associated cofactors. Different preparations of streptavidin show considerable heterogeneity at both the amino- and carboxy-termini of each subunit polypeptide due, to proteolysis during biosynthesis and secretion. Monomeric subunits of streptavidin are synthesized as 183 amino acid prepeptides. During secretion by Streptomyces sp., a 24 amino acid leader sequence is cleaved from these polypeptides resulting in newly secreted monomers of 159 amino acids (Agaraña et al, 1986). Upon longer incubations in culture these monomers are progressively cleaved to "core" subunits containing 125-127 amino acids (Bayer et al, 1989, Hendrickson et al, 1989). Preparations of streptavidin are relatively stable over a wide pH range, and extremely heat stable, requiring up to 20 minutes at 100C in 0.2% SDS to dissociate the subunits (Bayer et al, 1986). Strong chaotropic agents such as 6 M urea have been reported to dissociate the streptavidin tetramer into dimers (Sano and Cantor, 1990). These dimers appear to be stable in urea without appearance of monomers. Applications: Suitable for use in Enzyme conjugates and complexes, Southern Blots, DNA and RNA analysis, Western Blots and Protein Purification. Other applications not tested. Binding Properties: Unproteolyzed and proteolyzed preparations of streptavidin appear to bind biotin with equal affinity. The most highly proteolyzed tetramers may bind over 16 micrograms of d-biotin per milligram of protein. Bayer (1989) reports that biotinylated enzymes bind most effectively to truncated streptavidin in ELISA-type assays. ,   , Dissociation Constant:, The dissociation constant for biotin is approximately 10-15M (Chaiet et al, 1964). The formation, of the streptavidin-biotin complex is stable over wide pH and temperature ranges. The complex is generally disrupted only by conditions which lead to irreversible denaturation of the protein., Analogs of biotin such as 2-imino-biotin bind reversibly to the protein with complex formation at, high pH (>9.5) and dissociation at low pH (<4) (Bayer et al, 1986, Hofmann et al, 1980). Homogeneity: The streptavidin gene product is subject to cleavage by endogenous proteases during, fermentation. In some instances this can result in a heterogeneous product. The crude material may be treated with exogenous proteases during purification to reduce this heterogeneity. Source: Streptomyces avidinii, CAS No: 9013-20-1, Purity: Single band (SDS-PAGE), Form: Supplied as a lyophilized powder in sodium chloride. Specific Activity: >14U/mg protein, Molecular Weight: 55kD (4 similar polypeptide chains), Extinction Coefficient: 32 [1%, 280 nm] (Suter et al, 1988) , Dissociation Constant for Biotin: ~10-15M, Protein Content: ~0.9mg protein/mg lyophilate, Storage and Stability: Lyophilized powder may be stored at -20°C. Stable for 12 months afterr receipt at -20°C. Reconstitute with sterile buffer or ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. Country of Origin: USA