Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium.
A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D001710:Biotin / Q000031:analogs & derivatives; D003470:Culture Media / Q000032:analysis; D005374:Filtration; D007457:Iodine Radioisotopes; D008970:Molecular Weight; D019809:Streptavidin; D013302:Streptomyces / Q000254:growth & development*
D001426:Bacterial Proteins; D003470:Culture Media; D007457:Iodine Radioisotopes; D001710:Biotin; D019809:Streptavidin; C028072:2-iminobiotin