Yeast cell surface display of proteins and uses thereof
View Patent ↗The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 10 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components.
1. A fusion protein displayed on a yeast cell surface, the amino acid sequence of said fusion protein comprising a polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface, wherein said amino acid sequence further includes a first epitope tag sequence between said Aga2p and polypeptide sequence wherein said amino acid sequence further includes a second epitope tag sequence joined to the C-terminus of said polypeptide sequence.
2. The fusion protein of claim 1 wherein a label is bound to said second epitope tag.
3. A fusion protein displayed on a yeast cell surface, the amino acid sequence of said fusion protein consisting of a polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface wherein a label is bound to said polypeptide.
4. A fusion protein displayed on a yeast cell surface, the amino acid sequence of said fusion protein consisting of a polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface wherein said polypeptide is a ligand binding polypeptide wherein said ligand binding polypeptide is chosen from the group consisting of functional antibodies and cell surface receptors and fragments thereof wherein said polypeptide is an anti-T cell receptor or a fragment thereof.
5. A fusion protein displayed on a yeast cell surface, the amino acid sequence of said fusion protein consisting of a polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface wherein said polypeptide is a ligand binding polypeptide wherein said ligand binding polypeptide is chosen from the group consisting of functional antibodies and cell surface receptors and fragments thereof wherein said antibody or fragment thereof is an anti-T cell fragment chosen from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
6. A fusion protein displayed on a yeast cell surface, the amino acid sequence of said fusion protein consisting of a polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface wherein said polypeptide is a ligand binding polypeptide wherein said ligand binding polypeptide is chosen from the group consisting of functional antibodies and cell surface receptors and fragments thereof wherein said polypeptide is a T cell receptor or fragment thereof.
7. The fusion protein of claim 6 wherein said T cell receptor or fragment thereof has been mutagenized.
8. The fusion protein of claim 7 wherein said mutant T cell receptor or fragment thereof is SEQ ID NO:24.
9. A yeast cell displaying a fusion protein having an amino acid sequence consisting of a ligand binding polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface wherein said polypeptide sequence is a mutant of a related wild-type polypeptide.
10. In a yeast cell displaying a fusion protein, the improvement comprising: said fusion protein containing a mutant ligand binding protein joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the mutant ligand binding protein being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface.
11. In a yeast cell displaying a fusion protein, the improvement comprising: said fusion protein containing a mutated T cell binding protein joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the T cell binding protein sequence being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface.
12. In a yeast cell displaying a fusion protein, the improvement comprising: said fusion protein containing a mutated antibody or fragment thereof capable of antigen-specific binding joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the antibody or fragment being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface.