Yeast cell surface display of proteins and uses thereof

• US 6,696,251 B1
• Filed: 11/28/2000
• Issued: 02/24/2004
• 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: Kranz, David M., Wittrup, K. Dane, Boder, Eric T., Kieke, Michele
• Primary Examiner(s): Guzo, David
• Application Number:
  US09/724,108
• Time in Patent Office: 1,183 Days
• Field of Search: 435/320.1, 435/4, 435/6, 435/7.1, 435/7.2, 435/29, 435/471, 435/483, 435/69.1
• US Class Current: 435/6.14
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• 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. In 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 method of displaying more than one different polypeptide on the surface of yeast cells, comprising:

transforming yeast cells with vectors which express proteins to be displayed fused to yeast cell wall protens, wherein said proteins have 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.

2. The method of claim 1, wherein said polypeptides are one or more selected from the group consisting of those polypeptides of an organism which are displayable.

3. The method of claim 1, wherein at least one protein to be displayed is a T cell receptor or fragment thereof.

4. The method of claim 1, wherein said amino acid sequence further includes a first epitope tag sequence between said Aga2p and polypeptide sequence.

5. The method of claim 1, wherein said amino acid sequence further includes a second epitope tag sequence joined to the C-terminus of said polypeptide sequence.

6. The method of claim 1, wherein a label is bound to at least one ligand binding polypeptide sequence.

7. The method of claim 1, wherein at least one polypeptide is capable of binding a specific ligand.

8. The method of claim 1, wherein at least one polypeptide sequence is a mutant of a related wild-type polypeptide.

9. The method of claim 1, wherein said yeast strain is of a genus selected from the group consisting of:

Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia and Candida.

10. The method of claim 1, wherein said vectors comprise expression cassettes, each having the structure GAL 1-10 promoter.

11. The method of claim 1, wherein said ligand binding polypeptide sequence is selected from the group consisting of functional antibodies and cell surface receptors and fragments thereof.

12. The method of claim 2, wherein said organism is a mammal.

13. The method of claim 3, wherein the T cell receptor or fragment thereof is a single chain T cell receptor.

14. The method of claim 3, wherein the T cell receptor or fragment thereof has been mutagenized.

15. The method of claim 5, wherein a label is bound to said second epitope tag.

16. The method of claim 11 wherein said ligand binding polypeptide sequence is an anti-T cell receptor or a fragment thereof.

17. the method of claim 16 wherein said anti-T cell receptor or fragment thereof is selected from the group consisting of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

18. The method of claim 10 wherein at least one epitope tag is chosen from group consistng of SEQ ID NO:

1;

SEQ ID NO;

2, SEQ ID NO;

3;

SEQ ID NO;

4;

SEQ ID NO;

5;

SEQ ID NO;

6;

SEQ ID NO;

7;

SEQ ID NO;

8; and

SEQ ID NO;

9.

19. The method of claim 10 wherein said ligand binding polypeptide is an antibody or fragment thereof capable of antigen-specific binding.

20. The method of claim 10 wherein at least one polypeptide is a mutant polypeptide.

21. The method of claim 18 wherein at least one epitope tag is SEQ ID NO:

1.

22. The method of claim 18 wherein at least one epitope tag is SEQ ID NO:

2.

23. The method of claim 18 wherein the first epitope tag is SEQ ID NO:

1 and the second epitope tag is SEQ ID NO;

2.

24. The method of claim 20 wherein at least one ligand binding polypeptide is a receptor or fragment thereof.

25. The method of claim 19 wherein at least one 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.

26. The method of claim 19 wherein at least one antibody or fragment thereof is a single chain antibody.

27. The method of claim 24 wherein said receptor or fragrent thereof is a cell surface receptor or fragment thereof.

28. The method of claim 27 wherein said cell surface receptor or fragment thereof is a T cell receptor or fragment thereof.

29. The method of claim 27 wherein said cell surface receptor or fragment thereof is a mutant T cell receptor or fragment thereof.

30. The method of claim 28 wherein said T cell receptoror fragment thereof is SEQ ID NO:

24.