The strong biotin-streptavidin interaction limits the application of streptavidin as a reversible affinity matrix for purification of biotinylated biomolecules. To address this concern, a series of single, double and triple streptavidin muteins with different affinities to biotin were designed. The strategy involves mutating one to three strategically positioned residues (Ser45, Thr90 and Asp128) that interact with biotin and other framework structure maintaining residues of streptavidin. The muteins were produced in soluble forms via secretion from Bacillus subtilis. The impact of individual residues on the overall structure of streptavidin is reflected by the formation of monomeric streptavidin to different extents. Of the three targeted residues, Asp128 has the most dramatic effect (D128>T90>S45). Conversion of all three targeted residues to alanine results in a soluble biotin binding mutein that exists 100% in the monomeric state. Both wild-type and mutated (monomeric and tetrameric) streptavidin proteins were purified and their kinetic parameters (on- and off-rates) were determined using a BIAcore biosensor with biotin-conjugated bovine serum albumin immobilized to the sensor chip. This series of muteins shows a wide spectrum of affinity towards biotin (Kd from 10-6 to 10-11 M). Some of them have the potential to serve as reversible biotin binding agents.