Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium

Abstract

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 × g for 60 min. The absorbancy coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 ± 0.1806 for 1% solution. The protein appeared to be >90% homogenous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules <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°C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.