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...

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Bibliographic Details
Published inBio/technology (New York, N.Y. 1983) Vol. 11; no. 2; pp. 201 - 206
Main Authors Sano, T, Smith, C L, Cantor, C R
Format Journal Article
LanguageEnglish
Published United States 01.02.1993
Subjects
Amino Acid Sequence
Animals
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Base Sequence
Biotin - chemistry
Cell Membrane - chemistry
Cloning, Molecular
Cysteine - chemistry
Cysteine - genetics
DNA, Recombinant
Escherichia coli
Fluoresceins - chemistry
Genetic Vectors
Methylation
Mice
Molecular Sequence Data
Streptavidin
Sulfhydryl Compounds - chemistry
Tumor Cells, Cultured
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Summary: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.
ISSN:0733-222X
1087-0156
1546-1696
DOI:10.1038/nbt0293-201