(571n) Functional Expression of Streptavidin on Yeast Surface

Many proteins in nature form transient and permanent complexes with other proteins. With a few exceptions, however, the majority of the proteins successfully engineered using display technologies are single chain proteins that are immobilized on the exterior matrix of a cell or a phage particle. We are developing a general strategy for displaying proteins of quaternary structure on yeast surface. An ability to display multi-chain protein complexes would provide an important tool in the study of protein quaternary structure. To that end, we have displayed the bacterial protein streptavidin on yeast surface. Streptavidin is an obligate tetramer consisting of four identical chains and is used widely in biotechnology for its high affinity for the biotin molecule. Because the formation of a tetramer is essential for its function, testing for biotin binding is a highly sensitive assay for monitoring its quaternary structure. When we co-express anchored and soluble streptavidin monomers within the same cell, a functional streptavidin tetramer appears on yeast surface that binds biotin with high affinity. In contrast, expressing an anchored construct alone results in streptavidin that fails to bind biotin. Furthermore, adding biotin during protein synthesis substantially increased biotin binding, suggesting that biotin serves as a molecular chaperone during protein folding. The yeast cells displaying functional streptavidin on the surface can be used for biotechnological applications. To demonstrate this potential, we have immobilized biotinylated anti-his antibody on streptavidin expressing yeast, and used it to purify a protein from bacteria. Our study expands the potential application of yeast display to complex structures comprising multiple chains, and will help engineer other quaternary structures.