Expression and Purification of Recombinant Streptavidin Containing Chimeric Proteins

Takeshi Sano, Cassandra L. Smith, and Charles R. Cantor 1. Introduction

Streptavidm, a protein produced by Streptomyces avidinii, binds a water-soluble vitamin, D-biotin (vitamin H), with remarkably high affinity (1,2) The dissociation constant of the streptavidin-biotin complex is approx 10~l5M; the binding of streptavidm to biotin is one of the strongest noncovalent interactions found in biological systems. The extremely tight and specific biotin-bmding ability of streptavidm has made this protein a very powerful biological tool for a variety of biological and biomedical analyses (3,4) The ability of biotin to be incorporated easily into various biological materials has also expanded the application of the streptavidin-biotin technology to a wider range of biological systems

Several years ago, the streptavidin gene was cloned from a genomic library of S avidinii (5). The isolation of the streptavidm gene allowed the development of an efficient bacterial expression system for streptavidm (6), which is based on the bacteriophage T7 expression system (7) The establishment of the expression and purification methods for recombinant streptavidin allowed the design and production of streptavidin-contammg chimeric proteins. Such streptavidin-containing chimeras, if successfully produced, should have extremely tight and specific biotm-binding ability derived from the streptavidin moiety; this should provide the fused partner protein with highly specific, strong biological recognition capability that would allow, for example, specific conjugation, labeling, and targeting of the fused partner protein to any biological material containing biotin. Thus, such streptavidin-containing chimeras could serve as new, powerful biological tools in a variety of molecular

From Methods in Molecular Biology, vol 63 Recombinant Protein Protocols

Detection and Isolation Edited by R Tuan Humana Press Inc , Totowa, NJ

and cellular analyses, thereby further expanding the efficacy and versatility of the streptavidin-biotin technology.

This article describes general methods for expressing recombinant streptavidin-partner protein chimeras in Escherichia coh and purifying the expressed chimeric proteins. Although these methods have been used successfully for a few streptavidin-contaimng chimera constructs, some conditions and procedures may need to be modified or optimized for a particular partner protein fused to streptavidin to maximize the production of functional chimera molecules

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