[23] The S·tag fusion system for protein purification

This chapter discusses protein purification using S. tag fusion system. The S. Tag fusion system uniquely combines small tag size, antibody like ligand-binding specificity, and the ability to confer an easily measured enzymatic activity to fusion proteins. The S-protein ligand is also small, relatively inexpensive, and can be used in a variety of formats to enable many applications with a single tagging system. Virtually every technique used with antibodies and their epitope tags can be applied to the S-protein: S. Tag interaction, including the blotting and purification methods, fusion protein immobilization, affinity capture of interacting molecules, and the use of fluorophore-labeled S-protein for in situ affinity localization and cell sorting. The reconstitution of enzymatic activity by the simple addition of S-protein to any S.Tag fusion protein provides the platform for the development of novel assays. In particular, the discovery of a hypersensitive fluorogenic substrate for RNase A makes assays of S. Tag fusion proteins amenable to automation, and thus useful in a variety of high-throughput screening applications.