Yeast Display Systems

• US 20110275535A1
• Filed: 12/14/2009
• Published: 11/10/2011
• Est. Priority Date: 12/16/2008
• Status: Abandoned Application

• Current Assignee: Novartis Ag
• Original Assignee: Novartis Ag
• Inventors: Loew, Andreas
• Application Number:
  US13/139,332
• Publication Number:
  US 20110275535A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C07K 2319/912 containing a GPI (phosphatidyl-inositol glycane) anchor
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• 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: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention relates to the field of protein display libraries and library screening. In preferred embodiments, the present invention provides a three component system for display comprising a cell surface molecule, an adapter molecule and a display molecule.

Claims

1. A library of host cells, wherein each host cell comprising:

(a) a cell surface molecule attached to the surface of the cell,

(b) an adapter molecule comprising a first binding site and a second binding site, and

(c) a display molecule comprising a modified polypeptide;

wherein the first binding site binds specifically to the cell surface molecule and cannot bind to the display molecule and the second binding site binds specifically to the display molecule and cannot bind to the cell surface molecule, and wherein the adapter molecule is not a component of the modified polypeptide.

2. The host cells of claim 1, further comprising a plurality of display molecules.

3. The host cells of claim 1, wherein the host cell surface molecule is covalently linked to the first binding site.

4. The host cells of claim 1, wherein the host cell surface molecule is covalently linked to the first binding site through a disulfide bond.

5. The host cells of claim 1, wherein the host cell surface molecule comprises a first agglutinin that is Aga1p, and wherein the first binding site comprises a second agglutinin that is Aga2p.

7. The host cells of claim 1, wherein the host cell surface molecule is attached to the cell membrane via a GPI anchor.

10. The host cells of claim 1, wherein the second binding site is covalently linked to the display molecule.

11. The host cells of claim 1, wherein the second binding site is covalently linked to the display molecule through disulfide bonds.

12. The host cells of claim 1, wherein the second binding site comprises a PDZ domain of InaD.

14. The host cells of claim 12, wherein the display molecule comprises a C-terminal NorpA ligand.

15. The host cells of claim 1, wherein the display molecule comprises a PDZ domain of InaD.

17. The host cells of claim 15, wherein the second binding site comprises a C-terminal NorpA ligand.

18. The host cells of claim 1, wherein the modified polypeptide is selected from the group consisting of:

a scaffold protein, a signal transduction protein, an antibody, an immunoglobulin, an immunoadhesin, a receptor, a ligand, an oncoprotein, a transcription factor, an enzyme, and a fibronectin polypeptide.

19. The host cells of claim 18, wherein the display molecule is a fibronectin polypeptide.

20. The host cells of claim 19, wherein the fibronectin polypeptide comprises an F10 polypeptide.

21. The host cells of claim 1, wherein the display molecule comprises a secretion signal peptide.

22. The host cells of claim 21, wherein the secretion signal peptide comprises an MFalpha/HSA hybrid leader peptide, or an MFalpha leader peptide.

24. The host cells of claim 1, wherein expression of the display molecule is under the control of a first inducible promoter selected from the group consisting of an AOX1 promoter, a CUP 1 promoter, and a Gal 1 promoter.

27. The host cells of claim 1, wherein the expression of the adapter molecule is under the control of a second inducible promoter selected from the group consisting of an AOX1 promoter, a CUP 1 promoter, and a Gal 1 promoter.

30. The host cells of claim 1, wherein the host cell is a yeast cell.

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33. A method for displaying a modified polypeptide comprising:

(a) providing a host cell comprising a cell surface molecule attached to the surface of the cell and a first nucleic acid encoding a display polypeptide comprising a modified polypeptide,

(b) contacting the host cell with an adapter molecule comprising a first binding site and a second binding site under conditions wherein the first binding site binds to the cell surface molecule, and

(c) incubating the host cell under conditions wherein the host cell exports the display polypeptide outside the host cell under conditions wherein the second binding site binds to the display polypeptide,

wherein the first binding site binds specifically to the cell surface molecule and cannot bind to the display molecule and the second binding site binds specifically to the display polypeptide and cannot bind to the cell surface molecule, and wherein the adapter molecule is not a component of the modified polypeptide.

34. The method of claim 33, wherein the host cell displays at least 10², at least 10³, at least 10⁴, or at least 10⁵ modified polypeptides.

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64. A method for generating a host cell display library comprising:

introducing into a plurality of host cells a display library of first nucleic acids each encoding a display polypeptide comprising a modified polypeptide, wherein at least two of the introduced first nucleic acids encode different modified polypeptides,

wherein each host cell comprises a second nucleic acid which encodes a cell surface polypeptide and a third nucleic acid which encodes an adapter molecule comprising a first binding site and a second binding site, wherein the first binding site binds to the cell surface molecule but not the display polypeptide and the second binding site binds to the display polypeptide but not the cell surface molecule, and wherein the adapter molecule is not a component of the modified polypeptide.