A streptavidin mutant containing a cysteine stretch that facilitates production of a variety of specific streptavidin conjugates.
The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites. A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity. Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer. Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry. In this manner, S-[14C]carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared. These conjugates contain almost twenty [14C]carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive. More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.
Sano
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Department of Molecular and Cell Biology, University of California, Berkeley.
Smith
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CL
Cantor
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CR
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004274:DNA, Recombinant; D004926:Escherichia coli; D005452:Fluoresceins / Q000737:chemistry; D005822:Genetic Vectors; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D019809:Streptavidin; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D005452:Fluoresceins; D013438:Sulfhydryl Compounds; C015296:5-iodoacetamidofluorescein; D001710:Biotin; D019809:Streptavidin; D003545:Cysteine