Yeast cells expressing modified G proteins and methods of use therefor

• US 7,361,498 B2
• Filed: 10/15/2004
• Issued: 04/22/2008
• Est. Priority Date: 03/31/1993
• Status: Expired due to Fees

• Current Assignee: Cadus Technologies Incorporated
• Original Assignee: Cadus Technologies Incorporated
• Inventors: Broach, James R., Klein, Christine A., Manfredi, John P., Murphy, Andrew J. M., Paul, Jeremy I., Trueheart, Joshua, Fowlkes, Dana M.
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US10/967,087
• Publication Number:
  US 20050202403A1
• Time in Patent Office: 1,285 Days
• Field of Search: 435/252.3, 435/325, 536/23.1, 430/350, 530/350
• US Class Current: 435/252.3
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C07K 14/4722 G-proteins
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C40B 30/06 by measuring effects on living organisms, tissues or cells
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The present invention pertains to novel yeast cells which are useful for the expression of heterologous G protein coupled receptors. The yeast cells of the present invention can be used in screening assays which can be used to screen for modulators of G protein coupled receptors. Specifically, the invention provides novel yeast cells which express a heterologous G protein coupled receptor and mutant and/or chimeric G protein subunit molecules which serve to functionally integrate the heterologous into the pheromone signaling pathway of the yeast cell. The invention also provides for the expression of heterologous G protein coupled receptors which are functionally integrated into the yeast cell membrane using a yeast α factor leader sequence. Drug discovery assays using the subject yeast cells are also provided.

Claims

1. A yeast cell comprising a chimeric G protein subunit, said chimeric G protein subunit comprising a first polypeptide from a yeast G protein subunit and a second polypeptide from a heterologous G protein subunit, wherein said first polypeptide is selected from the group consisting of:

a polypeptide comprising about 40 amino acids from the amino terminus of yeast GPA1; and

a polypeptide from yeast STE 18.

2. The yeast cell of claim 1, wherein the cell is a Saccharomyces cerevisiae cell.

3. The yeast cell of claim 1, wherein said first polypeptide comprises about 40 amino acids from the amino terminus of yeast GPA1 and said second polypeptide is from a heterologous G protein α

subunit.

4. The yeast cell of claim 3, wherein said chimeric G protein subunit is selected from the group consisting of:

gpal (41)-Gi2;

gpal (41)-G16; and

gpal (41)-Gs.

5. The yeast cell of claim 4, wherein the Gα

i2, Gα

16, or Gα

s portion of said chimeric G protein subunit comprises an amino acid substitution compared to wild type Gi2, G16, or Gs.

6. The yeast cell of claim 1, wherein said heterologous G protein subunit is mammalian.

7. The yeast cell of claim 6, wherein said heterologous G protein subunit is human.

8. The yeast cell of claim 7, wherein at least one of said first and second polypeptides comprises a naturally occurring amino acid sequence.

9. The yeast cell of claim 7, wherein at least one of said first and second polypeptides comprises a non-naturally occurring amino acid sequence.

10. The yeast cell of claim 1, further comprising a heterologous G protein coupled receptor, which receptor is functionally integrated into the yeast cell.

11. The yeast cell of claim 10, wherein said chimeric G protein subunit demonstrates enhanced coupling to the heterologous G protein coupled receptor when compared to that demonstrated by an endogenous yeast G protein subunit.

12. The yeast cell of claim 10, wherein an endogenous yeast pheromone system receptor protein is not produced in functional form.

13. The yeast cell of claim 10, further comprising an indicator gene that produces a detectable signal upon functional coupling of the heterologous G protein coupled receptor to the G protein.

14. The yeast cell of claim 10, wherein said heterologous G protein coupled receptor is an orphan receptor.