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

• US 5,789,184 A
• Filed: 06/05/1995
• Issued: 08/04/1998
• Est. Priority Date: 03/31/1993
• Status: Expired due to Term

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Pharmaceutical Corporation
• Inventors: Trueheart, Joshua, Murphy, Andrew J., Klein, Christine, Paul, Jeremy, Fowlkes, Dana M., Broach, Jim, Manfredi, John
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Yucel, Irem
• Application Number:
  US08/464,531
• Time in Patent Office: 1,156 Days
• Field of Search: 435/4, 435/7.1, 435/64, 435/252.3, 435/320.1, 435/254.21, 435/254.2, 435/254.11
• US Class Current: 506/10
• 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 yeast cell having a pheromone system, which cell comprises

(a) a first heterologous gene encoding a heterologous surrogate of a yeast pheromone system protein, said surrogate being a kinase and performing in the pheromone system of the yeast cell a function naturally performed by the corresponding yeast pheromone system protein, and

(b) a second heterologous gene encoding a heterologous peptide, wherein said heterologous peptide modulates the interaction of said surrogate with said pheromone system in the yeast cell, and said modulation is a selectable or screenable event.

2. The yeast cell of claim 1 wherein the endogenous pheromone system protein is not produced in functional form.

3. The yeast cell of claim 1 wherein the heterologous peptide is secreted by the cell into the periplasmic space, from which it interacts with said surrogate.

4. The yeast cell of claim 3, wherein the heterologous peptide is expressed in the form of a precursor peptide comprising a cleavable leader peptide and a mature peptide, which leader peptide directs secretion of said heterologous peptide.

5. The yeast cell of claim 4 wherein the leader peptide corresponds to a leader peptide of the Saccharomyces cerevisiae α

factor or a-factor.

6. The yeast cell of claim 1 in which a wild-type pheromone of the yeast pheromone system is not secreted.

7. The yeast cell of claim 3 wherein the heterologous peptide is also expressed in a nonsecretory form.

8. The yeast cell of claim 1, wherein the cell is a mutant strain having a pheromone signal pathway that is desensitized at slower rate relative to the wild type strain under the same conditions of continuous stimulation of the pheromone signal pathway.

9. The yeast cell of claim 8 in which the endogenous SST2 gene is not functionally expressed.

10. The yeast cell of claim 1, in which the endogenous FAR1 gene is not functionally expressed.

11. The yeast cell of claim 1, further comprising a selectable marker that is activated by the pheromone signal pathway.

12. The yeast cell of claim 11, said selectable marker comprising a pheromone-responsive promoter which is substantially homologous with an endogenous pheromone-responsive promoter, operably linked to a foreign selectable gene.

13. The yeast cell of claim 12 wherein the selectable gene is an IGP dehydratase gene.

14. The yeast cell of claim 12 wherein the homologous wild-type promoter is the FUS1 promoter.

15. The yeast cell of claim 1 wherein the cells belong to the species Saccharomyces cerevisiae.

16. A yeast culture comprising a plurality of yeast cells according to claim 1, said yeast cells collectively expressing a peptide library.

17. A method of assaying a peptide for modulation of the activity of a non-yeast surrogate for a pheromone system protein which comprises providing yeast cells according to claim 1, which cells functionally express said heterologous surrogate and said heterologous peptide, and determining by detecting a change in said selectable or screenable event whether the pheromone signal pathway is activated or inhibited by the interaction of said surrogate and said peptide.

18. The method of claim 17 in which the cells comprise a pheromone-responsive selectable marker, and cells are selected for expression of a peptide having the desired activating or inhibiting effect.

19. The method of claim 17 in which the cells comprise a pheromone-responsive screenable marker, and cells are screened for expression of a peptide having the desired activating or inhibiting effect.

20. A method of assaying a peptide library for activity of a non-yeast pheromone system protein surrogate which comprises providing a yeast culture according to claim 16, whose cells each functionally express said surrogate and a peptide of said library, said culture collectively expressing the entire peptide library, and determining whether the pheromone signal pathway is activated or inhibited by said peptides in each of the cells of said culture.

46. The yeast cell of claim 1, in which the surrogate is a kinase selected from the group consisting of a mammalian MEK, MAPK, and MEKK.

47. The yeast cell of claim 1, in which the surrogate is a human MEK protein.

48. The yeast cell of claim 2, wherein an endogenous yeast cell kinase selected from the group consisting of MPK1, FUS1, KSS1, STE 11, STE 7, SPK1 and Byr1 is mutated.

21. A mixture of recombinant yeast cells, each cell of which comprises;

(i) a pheromone system generating a detectable signal;

(ii) an expressible gene construct encoding a heterologous surrogate of a yeast pheromone system protein, said surrogate being a kinase and performing in the pheromone system; and

(iii) an expressible gene construct encoding a heterologous peptide,

wherein collectively the mixture of cells express a library of said heterologous peptides, and modulation of the pheromone system by the heterologous peptide provides the detectable signal.

22. The recombinant cells of claim 21, wherein the yeast pheromone system protein is inactivated.

23. The recombinant cells of claim 21, wherein each cell further comprises a marker gene construct containing a marker gene in operative linkage with one or more transcriptional regulatory elements responsive to the pheromone system, expression of the marker gene providing the detectable signal.

24. The recombinant cells of claim 23, wherein the marker gene that gives rise to a detectable signal selected from the group consisting of:

β

galactosidase, alkaline phosphatase, horseradish peroxidase, exoglucanase, luciferase, and chloramphenicol acetyl transferase.

25. The recombinant cells of claim 23, wherein the marker gene that gives rise to a detectable signal is a HIS 3 gene.

26. The recombinant cells of claim 21, wherein the population of heterologous peptides includes at least 10³ different peptide sequences.

27. The recombinant cells of claim 21, wherein the population of heterologous peptides includes at least 10⁷ different peptide sequences.

28. The recombinant cells of claim 21, wherein the yeast cell is a Saccharomyces cell.

29. A mixture of recombinant yeast cells, each cell of which comprises:

(i) a pheromone system generating a detectable signal;

(ii) an expressible gene construct encoding a heterologous surrogate of a yeast pheromone system protein, said surrogate being a kinase and performing in the pheromone system; and

(iii) an expressible gene construct encoding a heterologous peptide, said heterologous peptide including a signal sequence for secretion into the periplasmic space,

wherein collectively the mixture of cells express a library of said heterologous peptides, and modulation of the pheromone system by the heterologous peptide provides the detectable signal.

30. The recombinant cells of claim 29, wherein the yeast pheromone system protein is inactivated.

31. The recombinant cells of claim 29, wherein each cell further comprises a marker gene construct containing a marker gene in operative linkage with one or more transcriptional regulatory elements responsive to the pheromone system, expression of the marker gene providing the detectable signal.

32. The recombinant cells of claim 31, wherein the marker gene encodes a gene product that gives rise to a detectable signal selected from the group consisting of:

β

galactosidase, alkaline phosphatase, horseradish peroxidase, exo glucanase, luciferase, and chloramphenicol acetyl transferase.

33. The recombinant cells of claim 31, wherein the marker gene that gives rise to a detectable signal is a HIS 3 gene.

34. The recombinant cells of claim 29, wherein the population of heterologous peptides includes at least 10³ different peptide sequences.

35. The recombinant cells of claim 29, wherein the population of heterologous peptides includes at least 10⁷ different peptide sequences.

36. The recombinant cells of claim 29, wherein the yeast cell is a Saccharomyces cell.

37. A method for identifying potential effectors of a yeast pheromone surrogate, comprising:

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

(a) a pheromone system generating a detectable signal;

(b) an expressible gene construct encoding a heterologous surrogate of a yeast protein of the pheromone system, said surrogate being a kinase and performing in the pheromone system; and

(c) an expressible gene construct encoding a heterologous peptide, wherein collectively the mixture of cells express a library of said heterologous peptides, and modulation of the pheromone system by the heterologous peptide provides the detectable signal; and

(ii) isolating cells from the mixture which exhibit the detection signal.

38. The method of claim 37, wherein the yeast pheromone system protein is inactivated.

39. The method of claim 37, wherein said heterologous peptide includes a signal sequence for secretion into the periplasmic space.

40. The method of claim 37, wherein each cell of the mixture further comprises a marker gene construct containing a marker gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the cell surface receptor protein, and wherein expression of the marker gene provides the detection signal.

41. The method of claim 40, wherein the marker gene encodes a gene product that gives rise to a detection signal selected from the group consisting of:

β

galactosidase, alkaline phosphatase, horseradish peroxidase, exo glucanase, luciferase, and chloramphenicol acetyl transferase.

42. The method of claim 40, wherein the marker gene that gives rise to a detectable signal is a HIS 3 gene.

43. The method of claim 38, wherein the population of heterologous peptides includes at least 10³ different peptide sequences.

44. The method of claim 38, wherein the population of heterologous peptides includes at least 10⁷ different peptide sequences.

45. The method of claim 37, wherein the yeast cell is a Saccharomyces cell.