Engineering soluble monomeric streptavidin with reversible biotin binding capability.
Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.
Wu
Sau-Ching
SC
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Wong
Sui-Lam
SL
D001412:Bacillus subtilis / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D012542:Scattering, Radiation; D012685:Sepharose / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D003432:Cross-Linking Reagents; D013912:Threonine; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin; D019286:Endopeptidase K; D014633:Valine