Arrestin biosensor

• US 8,883,486 B2
• Filed: 04/22/2011
• Issued: 11/11/2014
• Est. Priority Date: 05/04/2004
• Status: Active Grant

• Current Assignee: Universite De Montreal, L'
• Original Assignee: Universite De Montreal, L'
• Inventors: Bouvier, Michel, Beautrait, Alexandre, Charest, Pascale G., LeGouill, Christian, Laporte, Stephane Alain, Zimmerman, Brandon
• Primary Examiner(s): Shen, Bin
• Application Number:
  US13/092,667
• Publication Number:
  US 20110275134A1
• Time in Patent Office: 1,299 Days
• Field of Search: 435/287.1
• US Class Current: 435/287.1
• CPC Class Codes: G01N 2333/4704 Inhibitors; Supressors
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention relates to a novel biosensor. A resonance energy transfer (RET) biosensor comprising a beta(β)-arrestin tagged with a first and a second chromophore, wherein said first chromophore is a fluorophore and said second chromophore is a fluorophore or a bioluminophore is described.

Claims

1. A resonance energy transfer (RET) biosensor comprising an arrestin tagged with a first and a second chromophore, wherein said first chromophore is a fluorophore and said second chromophore is a fluorophore or a bioluminophore.

2. The biosensor according to claim 1, wherein said arrestin is β

-arrestin-1 (arrestin-2).

3. The biosensor according to claim 1, wherein said arrestin is β

-arrestin-2 (arrestin-3).

4. The biosensor according to claim 1, wherein said arrestin is arrestin-1 or arrestin-4.

5. The biosensor according to claim 1, wherein said second chromophore is a bioluminophore and the RET is bioluminescence resonance energy transfer (BRET).

6. The biosensor according to claim 5, wherein said bioluminophore is Renilla luciferase or a mutant form of Renilla luciferase.

7. The biosensor according to claim 6, wherein said bioluminophore is RlucII (A55T/C124A/M185V).

8. The biosensor according to claim 5, which is selected from the group consisting of:

Luc-β

-arr-YFP,

YFP-β

arr-Luc,

Luc-β

-arr(3A)-YFP,

Luc-β

-arr(IV)-YFP,

Luc-β

arr(R169E)-YFP,

mAmetrine-hβ

ar1-RlucI,

mAmetrine-hβ

arr2-RlucII,

CFP-hβ

arr1-RlucII,

CFP-hβ

arr2-RlucII,

GFP10-hβ

arr1-RlucII, and

GFP10-hβ

arr2-RlucII.

9. The biosensor according to claim 5, which is selected from the group consisting of:

RLucII-GDLRRALENSHASGYQACGTGS (SEQ ID NO;

5)-β

-arrestin-1-CLEDPRVPVAT (SEQ ID NO;

6)-YFP;

RLucII-GDLRRALENSHASGYQACGTGS (SEQ ID NO;

5)-β

-arrestin-2-CLEDPRVPVAT (SEQ ID NO;

6)-YFP;

RLucII-GDLRRALENSHASGYQACGTGS (SEQ ID NO;

5)-β

-arrestin R169E-CLEDPRVPVAT (SEQ ID NO;

6)-YFP;

GFP10-GSAGT (SEQ ID NO;

7)-β

-arrestin-1-KLPAT (SEQ ID NO;

8)-RlucII;

GFP10-GSAGT (SEQ ID NO;

7)-β

-arrestin-2-KLPAT (SEQ ID NO;

8)-RlucII;

GFP10-GSAGT (SEQ ID NO;

7)-β

-arrestin-R169E-KLPAT (SEQ ID NO;

8)-RlucII;

CFP-GSAGT (SEQ ID NO;

7)-β

-arrestin-1-KLPAT (SEQ ID NO;

8)-RlucII;

CFP-GSAGT (SEQ ID NO;

7)-β

-arrestin-2-KLPAT (SEQ ID NO;

8)-RlucII;

CFP-GSAGT (SEQ ID NO;

7)-β

-arrestin-R169E-KLPAT (SEQ ID NO;

8)-RlucII;

mAmetrine-GSAGT (SEQ ID NO;

7)-β

-arrestin-1-KLPAT (SEQ ID NO;

8)-RlucII;

mAmetrine-GSAGT (SEQ ID NO;

7)-β

-arrestin-2-KLPAT (SEQ ID NO;

8)-RlucII; and

mAmetrine-GSAGT (SEQ ID NO;

7)-β

-arrestin R169E-KLPAT (SEQ ID NO;

8)-RlucII.

10. The biosensor according to claim 1, wherein said first fluorophore is a fluorescent protein.

11. The biosensor according to claim 10, wherein said fluorescent protein is green fluorescent protein or a variant thereof.

12. The biosensor according to claim 10, wherein said fluorescent protein is yellow fluorescent protein (YFP), mAmetrine, cyan fluorescent protein (CFP), green fluorescent protein-2 (GFP-2), or green fluorescent protein-10 (GFP10).

13. The biosensor according to claim 1, wherein the first and second chromophores are independently attached to different positions on the arrestin, such positions being at the N-terminus, at the C-terminus, or at a position between the C- and N-termini of the arrestin.

14. The biosensor according to claim 1, wherein both said first and said second chromophores are fluorophores and the RET is fluorescence resonance energy transfer (FRET).

15. The biosensor according to claim 14, wherein the first chromophore is CFP or a variant thereof, and the second chromophore is a YFP or a variant thereof.

16. The biosensor according to claim 15, wherein the YFP is a non-circularly permuted sYFP2 or a circularly permuted sYFP2.

17. The biosensor according to claim 1, wherein the first chromophore, the second chromophore, or both the first and second chromophores are independently attached to the arrestin via a linker.

18. The biosensor according to claim 17, wherein the linker is a peptide linker.

19. The biosensor according to claim 18, wherein the peptide linker is 2 to 30 residues in length.

20. The biosensor according to claim 19, wherein the peptide linker is selected from the group consisting of GDLRRALENSHASAGYQACGTGS (SEQ ID NO:

5), CLEDPRVPVAT (SEQ ID NO;

6), GSAGT (SEQ ID NO;

7), KLPAT (SEQ ID NO;

8), GSAGTGSAGTGSAGT (SEQ ID NO;

9), KLPATKLPATKLPAT (SEQ ID NO;

10), and REAAAREAAAREAAAR (SEQ ID NO;

11).

21. The biosensor according to claim 19, wherein the linker is attached to the arrestin at the C-terminus, at the N-terminus, to a position between the C and N-termini, or to a combination thereof.