Yeast cells engineered to produce pheromone system protein surrogates and uses therefor

• US 7,105,309 B2
• Filed: 10/01/2002
• Issued: 09/12/2006
• Est. Priority Date: 03/31/1993
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Murphy, Andrew J. M., Trueheart, Joshua, Broach, James R., Klein, Christine A., Fowlkes, Dana M., Manfredi, John P., Paul, Jeremy I.
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US10/263,341
• Publication Number:
  US 20030203417A1
• Time in Patent Office: 1,442 Days
• Field of Search: 435/7.31, 435/254.11, 4352542- 21
• US Class Current: 435/7.31
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

Yeast cells are engineered to express both a surrogate of a pheromone system protein (e.g., enzymes involved in maturation of α-factor, transporters of a-factor, pheromone receptors, etc.) and a potential peptide modulator of the surrogate, in such a manner that the inhibition or activation of the surrogate affects a screenable or selectable trait of the yeast cells. Various additional features improve the signal-to-noise ratio of the screening/selection system.

Claims

1. A mixture of recombinant yeast cells, each cell of which has a cell membrane and each cell of which comprises:

(a) a heterologous receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the receptor expressed in the cell membrane;

wherein collectively the mixture of cells expresses a library of said heterologous polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the receptor by a heterologous polypeptide within the library provides a detectable signal.

2. The yeast cells of claim 1, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

3. The yeast cells of claim 2, wherein the signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

4. The yeast cells of claim 1, which belong to the species Saccharomyces cerevisiae.

5. The yeast cells of claim 1, wherein the library of heterologous polypeptides includes more than 1000 different polypeptide sequences.

6. A method for identifying a modulator of a heterologous receptor protein expressed by a yeast cell, comprising:

(i) providing a mixture of recombinant yeast cells, each cell of which has a cell membrane and comprises

(a) a heterologous receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

(b) a heterologous test polypeptide, wherein the heterologous test polypeptide is transported to a location allowing interaction with the extracellular region of the receptor expressed in the cell membrane;

 

wherein collectively the mixture of cells expresses a library of said heterologous test polypeptides, said library being expressible at a sufficient level such that modulation of the signal transduction activity of the receptor by a heterologous test polypeptide within the library provides a detectable signal;

(ii) allowing cells within the mixture to generate a detectable signal; and

(iii) identifying the heterologous test polypeptide as a modulator of the heterologous receptor protein expressed by the yeast cell.

7. The method of claim 6, wherein the heterologous receptor is a receptor that acts as a surrogate for an endogenous yeast pheromone receptor in a pheromone system pathway of the yeast cells.

8. The method of claim 7, wherein the yeast cells are a mutant strain of yeast cells having a pheromone system pathway that is desensitized at slower rate relative to a wild type strain under the same conditions of continuous stimulation of the pheromone system pathway.

9. The method of claim 7, wherein a gene encoding the endogenous yeast pheromone receptor is not functionally expressed in the yeast cells.

10. The method claim 7, wherein the yeast cells have a ste2 or ste3 mutation.

11. The method of claim 7, wherein an endogenous pheromone gene is not functionally expressed in the yeast cells.

12. The method of claim 7, wherein an endogenous FAR1 gene is not functionally expressed in the yeast cells.

13. The method of claim 7, wherein an endogenous SST2 gene is not functionally expressed in the yeast cells.

14. The method of claim 7, wherein the heterologus polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

15. The method of claim 14, wherein the signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

16. The method of claim 6, wherein the yeast cells belong to the species Saccharomyces cerevisiae.

17. The method of claim 6, wherein the library of heterologous polypeptides includes more than 1000 different polypeptide sequences.

18. A recombinant yeast cell which has a cell membrane and which comprises:

(a) a heterologous receptor expressed in the cell membrane of said cell such that signal transduction activity via said receptor is modulated by interaction of an extracellular region of the receptor with an extracellular signal; and

(b) a heterologous polypeptide, wherein the heterologous polypeptide is transported to a location allowing interaction with the extracellular region of the receptor expressed in the cell membrane;

wherein modulation of the signal transduction activity of the receptor by the heterologous polypeptide provides a detectable signal.

19. The yeast cell of claim 18, wherein the heterologous polypeptide includes a signal sequence that facilitates transport of the polypeptide to a location allowing interaction with the extracellular region of the receptor.

20. The yeast cell of claim 19, wherein the signal sequence corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

21. The yeast cell of claim 18, which belongs to the species Saccharomyces cerevisiae.