A Streptavidin Mutant Containing a Cysteine Stretch That Facilitates Production of a Variety of Specific Streptavidin Conjugates

Abstract

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.