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Affinity Reagents Based on Novel Molecular Scaffolds

Cheryl Baird, Principal Investigator

Schematic of the scaffold (Top7) displayed on the surface of yeast.
Schematic of the Top7 scaffold displayed on the surface of yeast. The Top7 scaffold is expressed as a protein fusion to the Aga2p mating agglutinin protein. The fusion protein is tethered to the yeast cell wall via disulfide bridges between the Aga2p protein and the Aga1p protein (which is covalently attached to the cell wall). The fusion protein also contains N-terminal HA and C-terminal c-myc epitope tags for detection of the displayed fusion. Yeast that display functional scaffolds that bind ligand can be identified and isolated by differential fluorescent staining of the scaffold and ligand. Click for a larger version.

The Affinity Reagents Based on Novel Molecular Scaffolds project team is developing a chemically robust, protein-based affinity reagent. This reagent will have applications to biodetection technologies, including protein microarrays, traditional immunoassays, molecular diagnostics, and biosensors for detecting biowarfare agents.

The scaffold is based on the protein Top7. Top7 was designed de novo in the laboratory of Dr. David Baker at the University of Washington to have a unique structure and high stability. We are testing the efficacy of using Top7 as a molecular scaffold to generate novel affinity reagents. Currently, yeast surface display selection methods and libraries are being developed to generate Top7 variants with affinity for select protein targets.

The protein, Top7, was designed de novo to have a unique structure and high stability.
(A) Sequence of Top7 depicting two-dimensional folding structure. Residues in blue boxes are found in helices, magenta hexagons in sheets, and green circles in loops or turns. (B) Ribbon diagram of Top7 based on the 2.5 Å crystal structure.
Yeast Displaying Top 7
Yeast displaying Top7 are identified via flow cytometry.

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