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J. Biol. Chem., Vol. 280, Issue 24, 23225-23231, June 17, 2005

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Engineering Soluble Monomeric Streptavidin with Reversible Biotin Binding Capability^*

Sau-Ching Wu and Sui-Lam Wong

From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

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.

Received for publication, February 15, 2005 , and in revised form, April 12, 2005.

^* This work was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada and a short term project grant from the University of Calgary. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1, S2, and S3.

To whom correspondence should be addressed: Dept. of Biological Sciences, Division of Cellular, Molecular and Microbial Biology, University of Calgary, 2500 University Dr., N. W. Calgary, Alberta T2N 1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong{at}ucalgary.ca.

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