Hirotaka Furukawa

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits.

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell‐surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C‐terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole‐cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo‐ and hetero‐oligomeric proteins, are displayed on the cell surface in an active form.

Preparation of yeast strains displaying IgG binding domain ZZ and enhanced green fluorescent protein for novel antigen detection systems

To develop novel immunofluorescence-labeling and antigen-detection systems, the ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G, and enhanced green fluorescent protein (EGFP) were displayed on the cell surface of Saccharomyces cerevisiae by cell-surface engineering using the C-terminus 318 amino acids of Flo1 protein. Two systems were constructed, one for co-display of ZZ and EGFP, and one for display of a fusion protein of the two. In both cases, two proteins on the cell surface successfully retain their activities.