Yeast cell surface display of proteins and uses thereof

• US 6,423,538 B1
• Filed: 11/28/2000
• Issued: 07/23/2002
• Est. Priority Date: 05/31/1996
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Keike, Michele, Kranz, David M., Wittrup, K. Dane, Boder, Eric T.
• Primary Examiner(s): Guzo, David
• Application Number:
  US09/724,297
• Time in Patent Office: 602 Days
• Field of Search: 435/320.1, 435/254.1, 435/254.11, 435/254.2, 435/6, 435/7.1, 435/7.31
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: G01N 2333/39 from yeasts
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

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 10⁴ protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. If effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

Claims

1. A vector comprising an expressible gene fusion which comprises a nucleic acid sequence coding for a yeast cell wall protein fused to a nucleic acid sequence coding for a protein or peptide to be tested for improved binding to a label wherein the yeast cell wall protein is attached to the yeast cell wall through disulfide bonds.

2. The vector of claim 1 wherein the yeast cell wall protein is the binding subunit of an agglutinin.

3. The vector of claim 1 wherein the protein or peptide to be tested is an antibody.

4. The vector of claim 1 wherein the protein or peptide to be tested is a single-chain antibody.

5. The vector of claim 1 wherein the protein or peptide to be tested is a T cell receptor.

6. The vector of claim 1 wherein the protein or peptide to be tested is a single chain T cell receptor.

7. The vector of claim 1 wherein the protein or peptide to be tested is a Fab fragment of an antibody.

8. The vector of claim 1 wherein the protein or peptide to be tested is a Fv fragment of an antibody.

9. The vector of claim 1 wherein the yeast cell wall protein is Aga2p.

10. A vector comprising a nucleic acid sequence coding for a yeast cell wall protein fused to a nucleic acid sequence coding for a protein or polypeptide or peptide to be displayed on the surface of a yeast cell wherein the protein or polypeptide or peptide to be displayed is fused to the C-terminus of the yeast cell wall protein.

11. The vector of claim 10 wherein the yeast cell wall protein is the binding subunit of an agglutinin.

12. The vector of claim 11 wherein the yeast cell wall protein is attached to the cell wall through disulfide bonds.

13. A library of vectors comprising an expressible gene fusion which comprises a nucleic acid sequence coding for a yeast cell wall protein fused to a member of a variegated population of nucleic acid sequences coding for a protein, polypeptide or peptide to be tested for improved binding to a label wherein the yeast cell wall protein is attached to the yeast cell wall through disulfide bonds.

14. A library of vectors comprising a nucleic acid coding for a yeast cell wall protein fused to a variegated population of nucleic acid sequences coding for a protein or polypeptide or peptide to be displayed on the surface of a yeast cell wherein the protein or polypeptide or peptide to be displayed is fused to the C-terminus of the yeast cell wall protein.

15. The vector of claim 14, wherein said Aga2p binds to Aga1p which binds to the yeast cell wall.