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J. Biol. Chem., Vol. 276, Issue 49, 46422-46428, December 7, 2001
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From the Department of Biological Sciences, Division of Cellular,
Molecular and Microbial Biology, University of Calgary, Alberta T2N
1N4, Canada
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 (Ser-45,
Thr-90, and Asp-128) 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,
Asp-128 has the most dramatic effect (Asp-128 > Thr-90 > Ser-45). 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 toward biotin
(Kd from 10
Development and Characterization of a Series of Soluble
Tetrameric and Monomeric Streptavidin Muteins with Differential Biotin
Binding Affinities*
,
6 to
1011 M). Some of them have the potential to
serve as reversible biotin binding agents.
*
This research was supported by a strategic grant from the
Natural Sciences and Engineering Research Council of Canada (NSERC).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: KS Avicenna, Edmonton Research Park, 1938-94th
St., Edmonton, Alberta, T6N 1J3, Canada.
§
Supported in part by summer studentships from Alberta Heritage
Foundation for Medical Research and NSERC.
¶
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@ucalgary.ca.
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