Immobilized nitro-avidin and nitro-streptavidin as reusable affinity matrices for application in avidin-biotin technology.
Chemically modified forms of egg-white avidin and bacterial streptavidin (termed nitro-avidin and nitro-streptavidin, respectively), in which the binding-site tyrosine was nitrated, were used for several biotechnological applications. The fundamental difference between nitro-avidin and the native protein is that interaction of the modified protein with biotin can be reversed under relatively mild conditions. Consequently, nitro-avidin affinity columns or immobilizing matrices can be reused. Three examples are given to demonstrate the possible uses of such columns: (a) biotinylated protein A was attached to a nitro-avidin affinity column, and immunoglobulin was purified directly from whole rabbit serum; (b) biotinylated transferrin was attached to a nitro-streptavidin column, and anti-transferrin was isolated directly from rabbit anti-serum; and (c) biotinylated beta-glucosidase was immobilized onto a nitro-avidin column and used as an enzyme reactor. In each example, the immobilized biotinylated probe could be released selectively from the column and recovered following its utilization. Reusable nitro-avidin thus provides an easy and attractive reversible form of avidin and thereby serves to expand the versatility of avidin-biotin technology.
Morag
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Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, 76100, Israel. bfbayer@wiccmail.weizmann.ac.il
Bayer
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EA
Wilchek
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D000818:Animals; D001360:Avidin / Q000031:analogs & derivatives* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001709:Biotechnology; D001710:Biotin / Q000031:analogs & derivatives*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D008024:Ligands; D009566:Nitrates / Q000737:chemistry; D011817:Rabbits; D019809:Streptavidin; D014168:Transferrin / Q000276:immunology; D014443:Tyrosine / Q000737:chemistry; D001617:beta-Glucosidase / Q000302:isolation & purification
D001426:Bacterial Proteins; D007074:Immunoglobulin G; D008024:Ligands; D009566:Nitrates; D014168:Transferrin; D001360:Avidin; D014443:Tyrosine; D001710:Biotin; D019809:Streptavidin; D001617:beta-Glucosidase