Permuted and nonpermuted luciferase biosensors

Patent metadata

Field	Value
Publication number	US-9359635-B2
Application number	US-73210507-A
Country	US
Kind code	B2
Filing date	Apr 2, 2007
Priority date	Apr 3, 2006
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

A modified luciferase protein which is a sensor for molecules including cAMP, cGMP, calcium, chelators thereof, kinases, or phosphatases is provided. Also provided is a circularly permuted anthozoan luciferase protein and a decapod crustacean luciferase protein, optionally containing one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest. Further provided is a modified anthozoan luciferase protein and a decapod crustacean luciferase protein containing an insertion of one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest.

First claim

What is claimed is:

1. An isolated polynucleotide comprising a nucleic acid sequence encoding a circularly permuted luciferase, the circularly permuted luciferase comprising a cyclic nucleotide binding site, which circularly permuted luciferase being permuted at a site or in a region which in a corresponding nonpermuted luciferase is tolerant to modification, and wherein luciferase activity of the circularly permuted luciferase is detectable, wherein the cyclic nucleotide binding site comprises at least a portion of a protein selected from the group consisting of exchange protein directly activated by cAMP (Epac), cyclic nucleotide gated ion channels, neuropathy target esterase, PKA regulatory type IIβ subunit, PKA regulatory type Iα subunit, catabolite activating protein, cGMP dependent protein kinase (GK), GAF regulatory region in phosphodiesterase, adenyl cyclases and FnlA, wherein the permutation is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue 102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353 to 408, residue 485 to 495, or residue 535 to 546 of a firefly luciferase of SEQ ID NO: 210, a region corresponding to residue 15 to 30, residue 112 to 122, residue 352 to 362, residue 371 to 384, residue 393 to 414, or residue 485 to 495 of a click beetle luciferase of SEQ ID NO: 3, or a region corresponding to residue 2 to 12, residue 26 to 47, residue 64 to 74, residue 85 to 116, residue 147 to 157, residue 223 to 234, or residue 301 to 311 of a Renilla luciferase of SEQ ID NO: 308.

2. The isolated polynucleotide of claim 1 wherein the circularly permuted luciferase further comprises a tag of at least one amino acid at the N-terminus, C-terminus, or both.

3. The isolated polynucleotide of claim 2 wherein the tag is a PEST sequence, a GST sequence, or a polyhistidine sequence.

4. The isolated polynucleotide of claim 1 wherein the circularly permuted luciferase further comprises a deletion of 1 to 30 residues of luciferase sequences at sequences corresponding to the N-terminus and/or C-terminus of the nonpermuted luciferase.

5. The isolated polynucleotide of claim 1 or 4 wherein the cyclic nucleotide binding site is at sequences corresponding to the N-terminus and/or C-terminus of the nonpermuted luciferase.

6. The isolated polynucleotide of claim 1 wherein the circularly permuted luciferase is a firefly or click beetle luciferase.

7. The polynucleotide of claim 1 wherein the luciferase is a Renilla luciferase.

8. The polynucleotide of claim 1 wherein the cyclic nucleotide binding site is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue 102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353 to 408, residue 485 to 495, or residue 535 to 546 of a firefly luciferase of SEQ ID NO: 210.

9. The polynucleotide of claim 1 wherein the cyclic nucleotide binding site is in a region corresponding to residue 15 to 30, residue 112 to 122, residue 352 to 362, residue 371 to 384, residue 393 to 414, or residue 485 to 495 of a click beetle luciferase of SEQ ID NO: 3.

10. The polynucleotide of claim 1 wherein the cyclic nucleotide binding site is in a region corresponding to residue 2 to 12, residue 26 to 47, residue 64 to 74, residue 85 to 116, residue 147 to 157, residue 223 to 234, or residue 301 to 311 of a Renilla luciferase of SEQ ID NO: 308.

11. The isolated polynucleotide of claim 1 wherein the cyclic nucleotide binding site binds cAMP.

12. The isolated polynucleotide of claim 1 wherein the cyclic nucleotide binding site binds cGMP.

13. A vector comprising the isolated polynucleotide of claim 1 .

14. An isolated host cell comprising the vector of claim 13 .

15. A circularly permuted luciferase encoded by the polynucleotide of claim 1 .

16. A method to detect or determine a cyclic nucleotide in a cell, comprising:
a) providing a mixture comprising the isolated host cell of claim 14 and reagents for a luminescence reaction; and
b) detecting or determining luminescence in the mixture, thereby detecting or determining the presence or amount of cyclic nucleotide in the isolated host cell.

17. A method to detect or determine a cyclic nucleotide in a sample, comprising:
a) providing a mixture comprising a sample suspected of having cyclic nucleotide, the circularly permuted luciferase of claim 15 , and reagents for a luminescence reaction; and
b) detecting or determining luminescence in the mixture.

18. The method of claim 16 or 17 wherein the luciferase is a firefly or click beetle luciferase.

19. The method of claim 16 or 17 wherein the luciferase is a Renilla luciferase.

20. The method of claim 16 or 17 wherein binding of cyclic nucleotide to the cyclic nucleotide binding site results in a change in luminescence.

21. The method of claim 20 wherein the binding results in an increase in luminescence.

22. The method of claim 17 wherein the sample comprises cells.

23. The method of claim 17 wherein the sample comprises a cell lysate or cell fraction.

24. A method to detect one or more modulators of a G protein coupled receptor, comprising:
a) providing a sample comprising one or more test agents, the isolated host cell of claim 14 and reagents for a luminescence reaction; and
b) detecting or determining luminescence in the sample.

25. A method to detect one or more modulators of a G protein coupled receptor, comprising:
a) providing a sample comprising one or more test agents, the circularly permuted luciferase of claim 15 , and reagents for a luminescence reaction; and
b) detecting or determining luminescence in the sample.

26. A method to detect one or more modulator of a G protein coupled receptor, comprising:
a) comparing luminescence from a first luminogenic reaction mixture comprising one or more test agents, the isolated host cell of claim 14 and reagents for a luminescence reaction to luminescence from a corresponding luminogenic reaction mixture that does not contain one or more test agents, but includes the isolated host cell of claim 14 and reagents for a luminescence reaction; and
b) detecting or determining whether the one or more test agents in the first luminogenic reaction mixture alter the luminescence in the first luminogenic reaction mixture relative to the corresponding luminogenic reaction mixture.

27. A method to detect one or more modulators of a G protein coupled receptor, comprising:
a) comparing luminescence from a first luminogenic reaction mixture comprising one or more test agents, the circularly permuted luciferase of claim 15 , and reagents for a luminescence reaction to luminescence from a corresponding luminogenic reaction mixture that does not contain the one or more test agents, but includes the circularly permuted luciferase of claim 15 and the reagents; and
b) detecting or determining whether the agents in the first luminogenic reaction mixture alter the luminescence in the first luminogenic reaction mixture relative to the corresponding luminogenic reaction mixture.

28. The polynucleotide of claim 1 , wherein the cyclic nucleotide binding site comprises at least a portion of a protein selected from the group consisting of exchange protein directly activated by cAMP (Epac)

Assignees

• Binkowski Brock
• Fan Frank
• Wigdal Susan
• Wood Keith V
• Wood Monika G
• Promega Corp

Inventors

• Binkowski Brock
• Fan Frank
• Wigdal Susan
• Wood Keith V
• Wood Monika G

Classifications

• G01N33/84

  involving inorganic compounds or pH · CPC title

• C12Y113/12007

  Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase · CPC title

• C12Q1/66 Primary

  involving luciferase · CPC title

• C12N9/0069 Primary

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12N9/0004

  Oxidoreductases (1.) · CPC title