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

• US 7,611,854 B2
• Filed: 06/18/2003
• Issued: 11/03/2009
• Est. Priority Date: 03/31/1993
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Trueheart, Joshua, Murphy, Andrew J., Klein, Christine, Paul, Jeremy, Broach, Jim, Manfredi, John, Fowlkes, Dana M.
• Primary Examiner(s): Ulm; John D
• Application Number:
  US10/600,003
• Publication Number:
  US 20040197840A1
• Time in Patent Office: 2,330 Days
• Field of Search: None
• 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 method of detecting ligand binding to a heterologous G protein coupled receptor comprising:

a) providing transformed yeast cells comprising a reporter gene under control of a pheromone-responsive promoter, a heterologous G protein-coupled receptor gene, each said gene being under the control of a separate promoter, a mutation in a SCG1/GPA1 gene, and a hybrid G.alpha. protein;

b) combining said cells with a compound to be tested; and

c) detecting if said compound binds to said heterologous G protein coupled receptor.

2. The method of claim 1, wherein said heterologous G protein coupled receptor is a mammalian G protein coupled receptor.

3. The method of claims 1 or 2, wherein said hybrid G.alpha. protein comprises yeast G.alpha. protein sequences and heterologous G.alpha. protein sequences.

4. The method of claims 1 or 2, wherein the reporter gene is selected from the group consisting of HIS3, G418r, URA3, LYS2, CAN1, Lacz, and CYH2 and the pheromone-responsive promoter is FUS 1.

5. The method of claim 4, wherein said reporter gene is selected from the group consisting of HIS3, G418r, URA3, LYS2, CAN1, and Lacz.

6. The method of claim 1, wherein said heterologous G protein-coupled receptor is selected from the group consisting of .beta.2 adrenergic receptor, .alpha.2 adrenergic receptor, 5HT 1-A receptor, muscarinic acetylcholine receptor, growth hormone releasing factor receptor, and somatostatin receptor.

7. The method of claim 1, wherein said heterologous G protein-coupled receptor gene encodes a receptor selected from the group consisting of .beta.2 adrenergic receptor, .alpha.2-adrenergic receptor, 5HT-1A receptor, muscarinic acetylcholine receptor, growth hormone releasing factor receptor, somatostatin receptor, cholecystokinin receptor, and adenosine receptor.

8. The method of claim 7, wherein said heterologous G protein-coupled receptor is selected from the group consisting of .beta.2 adrenergic receptor, .alpha.2 adrenergic receptor, 5HT 1-A receptor, muscarinic acetylcholine receptor, growth hormone releasing factor receptor, and somatostatin receptor.

9. The method of claims 1 or 2, wherein said transformed yeast cell further comprises a heterologous G.alpha. subunit.

10. The method of claim 9, wherein said heterologous G.alpha. subunit is selected from the group consisting of a Gs subunit, a Gi subunit, a Go subunit, a Gz subunit, a Gq subunit, a G11 subunit, and a G16 subunit.