Design, production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins.
To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.
Department of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4.
Mohammad Hassan
MH
Qureshi
Sui-Lam
SL
Wong
D001360:Avidin / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D019921:Biotinylation; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015195:Drug Design; D005822:Genetic Vectors; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D019809:Streptavidin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013816:Thermodynamics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; C049538:streptavidin-agarose; D001360:Avidin; D012685:Sepharose; D019809:Streptavidin