Engineered Chimeric Streptavidin Tetramers as Novel Tools for Bioseparations and Drug Delivery
- 1 November 1995
- journal article
- Published by Springer Nature in Nature Biotechnology
- Vol. 13 (11), 1198-1204
- https://doi.org/10.1038/nbt1195-1198
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 thiocyanateinduced 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 His127Cys 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, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose 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.Keywords
This publication has 24 references indexed in Scilit:
- The in vivo uses of streptavidin and biotin: a short progress reportNuclear Medicine Communications, 1994
- Pharmacokinetics and biodistribution of radiolabeled avidin, streptavidin and biotinNuclear Medicine and Biology, 1993
- A Streptavidin–Protein a Chimera that allows one–step production of a variety of Specific Antibody ConjugatesBio/Technology, 1991
- Monoclonal antibody pretargetting techniques for tumour localizationNuclear Medicine Communications, 1991
- Intraperitoneal radio‐localization of tumors pre‐targeted by biotinylated monoclonal antibodiesInternational Journal of Cancer, 1990
- [2] Introduction to avidin-biotin technologyMethods in Enzymology, 1990
- Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation.Proceedings of the National Academy of Sciences, 1989
- Structural Origins of High-Affinity Biotin Binding to StreptavidinScience, 1989
- In vivo labelling of biotinylated monoclonal antibodies by radioactive avidin: A strategy to increase tumor radiolocalizationInternational Journal of Cancer, 1988
- AvidinAdvances in protein chemistry, 1975