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Engineered Chimeric Streptavidin Tetramers as Novel Tools for Bioseparations and Drug Delivery

Bio/Technology volume 13, pages 1198–1204 (1995)Cite this article

Abstract

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanate-induced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/N49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the Hisl27Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trpl20Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 104 compared to WT streptavidin. The W120A biotin-binding affinity is sufficiently high (Ka ≈107 M−1) to immobilize the mutant on a biotin-agarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

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Authors and Affiliations

  1. Molecular Bioengineering Program, Center for Bioengineering, University of Washington, Box 357962, Seattle, WA, 98195

    Ashutosh Chilkoti, Cynthia J. Long & Patrick S. Stayton

  2. Chemical Methods and Separations Group, Chemical Sciences Department, Pacificfie Northwest Laboratory, Richland, WA, 99352

    Brenda L. Schwartz & Richard D. Smith

Authors
  1. Ashutosh Chilkoti
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  2. Brenda L. Schwartz
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  3. Richard D. Smith
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  4. Cynthia J. Long
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  5. Patrick S. Stayton
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Correspondence to Patrick S. Stayton.

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Chilkoti, A., Schwartz, B., Smith, R. et al. Engineered Chimeric Streptavidin Tetramers as Novel Tools for Bioseparations and Drug Delivery. Nat Biotechnol 13, 1198–1204 (1995). https://doi.org/10.1038/nbt1195-1198

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