Yeast cell surface display of proteins and uses thereof

• US 6,300,065 B1
• Filed: 08/26/1998
• Issued: 10/09/2001
• 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: Wittrup, K. Dane, Boder, Eric T., Shusta, Eric, Kieke, Michele C., Kranz, David M.
• Primary Examiner(s): Guzo, David
• Application Number:
  US09/140,084
• Time in Patent Office: 1,140 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/320.1, 435/7.2, 435/7.31, 435/29, 435/69.1, 435/325, 435/348, 435/252.3, 435/254.11, 435/254.1, 435/254.21, 435/254.22, 435/254.23
• US Class Current: 435/6.14
• 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 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• 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. 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. 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. As another embodiment, the selection method identifies proteins displayed at higher levels as proteins that are secreted at higher efficiency and that are more stable.

Claims

1. A method for selecting proteins with enhanced phenotypic properties relative to those of a wild-type of said protein, comprising the steps of:

transforming yeast cells with a vector expressing a protein to be tested fused to a yeast cell wall protein, wherein mutagenesis is used to a generate a variegated population of mutants of the protein to be tested;

contacting said yeast cells with a first label, wherein said first label associates with yeast expressing said protein having enhanced phenotypic properties and does not associate with yeast which do not express said protein having enhanced phenotypic properties;

isolating said yeast cells with which said first label is associated;

analyzing and comparing said phenotypic properties of said mutant protein expressed by yeast with phenotypic properties of said wild-type protein; and

selecting yeast cells exhibiting mutant proteins with enhanced phenotypic properties over the wild-type protein;

where enhanced phenotypic properties are one or more of surface expression level, stability, secretion levels and solubility.

2. The method of claim 1, wherein said protein to be tested is an antibody, Fab, Fv, or scFv antibody fragment.

3. The method of claim 1, wherein said protein to be tested is the ligand binding domain of a cell surface receptor.

4. The method of claim 1, wherein the protein to be tested is fused by its N terminus to the C terminus of said yeast cell wall protein.

5. The method of claim 1, wherein the yeast cell wall protein is an agglutinin.

6. The method of claim 1, wherein said yeast is of a genus selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida.

7. The method of claim 1, wherein said mutants of the protein to be tested are selected from the group consisting of single mutants and multiple mutants.

8. The method of claim 1, wherein said first label is selected from the group consisting of magnetic particles attached to a ligand for the protein to be and fluorescent labels.

9. The method of claim 1, wherein selection of mutated proteins of interest with enhanced phenotypic properties involves iterative cycles of said transforming and contacting steps.

10. The method of claim 1, further comprising the steps of:

contacting said yeast cells with a second label, wherein said vector used to transform said yeast cells contains means for expressing a polypeptide sequence fused to said protein to be tested to produce a fusion polypeptide and said second label associates with yeast cells expressing said fusion polypeptide and does not associate with yeast cells which do not express said fusion polypeptide;

enriching a transformed yeast population by quantitating said second label, wherein an occurrence of said second label is directly proportional to an abundance of said fusion polypeptides expressed on the cell surface; and

comparing said quantitation of said first label to said quantitation of said second label to determine surface expression levels of said protein to be tested.

11. The method of claim 1, further comprising the steps of:

cloning a gene encoding said selected mutant proteins into a vector adapted for expression in a eukaryotic cell; and

expressing said mutant protein in said eukaryotic cell, wherein said enhanced phenotype properties of said mutant protein are confirmed by comparing the properties of said enhanced phenotypic properties of said mutant protein with the properties of said wild-type protein.

12. The method of claim 1, further comprising the steps of:

cloning a gene encoding said selected mutant proteins into a vector adapted for expression in a prokaryote; and

expressing said mutant protein in said prokaryote, wherein said enhanced phenotypic properties of said mutant protein are confirmed by comparing the properties of said enhanced phenotypic properties of said mutant protein with the properties of said wild-type protein.

13. The method of claim 3, wherein said cell surface receptor is a T cell receptor.

14. The method of claim 10, wherein an increase in said surface expression levels of said mutant protein to be tested relative to the level of surface expression of the wild-type protein tested can be used to select for desirable phenotypic properties of said mutant protein, where said desired phenotypic properties include one or more of intracellular expression level, stability, secretion levels and solubility.

15. The method of claim 10, wherein said polypeptide portion of said fusion polypeptide recognized by said second label is an epitope tag.

16. The method of claim 10, wherein said first label and said second label are fluorescent labels.

17. The method of claim 11, wherein said eukaryotic cell is selected from the group consisting of mammalian, insect and yeast cells.