Protease-based biosensor

Patent metadata

Field	Value
Publication number	US-9791436-B2
Application number	US-201314427982-A
Country	US
Kind code	B2
Filing date	Sep 12, 2013
Priority date	Sep 12, 2012
Publication date	Oct 17, 2017
Grant date	Oct 17, 2017

Abstract

A biosensor molecule comprises: a protease amino acid sequence; at least one sensor comprising at least one sensor amino acid sequence which is responsive to at least one target molecule; and an inhibitor of the protease activity of said protease amino acid sequence; wherein the biosensor is switchable from a protease active to a protease inactive state, or from a protease inactive to a protease active state when said sensor responds to said target molecule. The biosensor protease may be a protease of a virus such as a Potyvirus or a Flavivirus wherein the inhibitor is an autoinhibitory peptide derived from the virus. The biosensor may respond to the target molecule allosterically or may be cleaved by a target protease molecule.

First claim

The invention claimed is:

1. A biosensor molecule comprising a protease amino acid sequence; at least one sensor comprising at least one sensor amino acid sequence which is responsive to at least one target molecule; and an inhibitor of the protease activity of said protease; wherein the biosensor is switchable from a protease active to a protease inactive state, or from a protease inactive to a protease active state when said sensor responds to said target molecule.

2. The biosensor of claim 1 , comprising (i) a single sensor comprising a single amino acid sequence responsive to a single target molecule; (ii) two sensors comprising respective amino acid sequences responsive to the same or different target molecule; (iii) a single protease amino acid sequence and a single inhibitor; and/or (iv) two different protease amino acid sequences and respective inhibitors.

3. The biosensor of claim 1 , wherein the or each sensor allosterically activates or inhibits the protease activity of the biosensor upon responding to the target molecule.

4. The biosensor of claim 3 , wherein the or each sensor allosterically releases inhibition of the or each protease by the inhibitor of the protease activity of said protease.

5. The biosensor of claim 1 , wherein the or each sensor comprises an affinity clamp; wherein the affinity clamp comprises a recognition domain; wherein the recognition domain is capable of binding one or more target molecules.

6. The biosensor of claim 5 , wherein the affinity clamp further comprises an enhancer domain.

7. The biosensor of claim 6 , wherein the recognition domain is a PDZ domain.

8. The biosensor of claim 6 , wherein the enhancer domain is a type III domain of fibronectin.

9. The biosensor of claim 1 , wherein the or each sensor comprises one or a plurality of epitopes that can be bound by an antibody target molecule, so that said sensor responds to said antibody target molecule.

10. The biosensor of claim 1 , wherein the or each sensor comprises an antibody which binds the target molecule.

11. The biosensor of claim 10 , which further comprises a molecule which binds the antibody.

12. The biosensor of claim 11 the molecule is a ZZ domain of Protein A which binds the antibody.

13. The biosensor of claim 1 , wherein the or each sensor amino acid sequence comprises a protease cleavage site cleavable by a protease target molecule to thereby at least partly release inhibition of the protease by the inhibitor and switch the biosensor from a protease active to a protease inactive state.

14. The biosensor of claim 1 , wherein the biosensor protease is a cysteine protease, a serine protease, an aspartate protease, a metalloprotease, a threonine protease, or a glutamic acid protease.

15. The biosensor of claim 14 , wherein the biosensor protease is derived or obtainable from a virus.

16. The biosensor of claim 15 , wherein the virus is a Potyvirus or a Flavivirus.

17. The biosensor of claim 16 , wherein the protease is an NIa protease.

18. The biosensor of claim 1 , wherein the inhibitor is a peptide.

19. The biosensor of claim 1 , which comprises first and second sensors responsive to the same or different target molecules.

20. The biosensor of claim 19 , which is a circularly permutated biosensor.

21. The biosensor of claim 1 further comprising an amplifier interacting domain for linking or coupling the biosensor to an amplifier molecule.

22. The biosensor of claim 1 comprising an amino acid sequence set forth in any one of SEQ ID NOS: 1-10.

23. An amplifier molecule operable with the biosensor molecule of claim 1 that comprises: (i) an amino acid sequence of a protease that is different to the protease(s) of the biosensor; (ii) an inhibitor of the protease of (i); and (iii) a linker amino acid sequence which comprises a cleavage site for the protease(s) of the biosensor.

24. The amplifier molecule of claim 23 which comprises a biosensor interacting domain for linking or coupling the amplifier molecule to the biosensor.

25. A composition or kit comprising the biosensor of claim 1 and a substrate.

26. The composition or kit of claim 25 , wherein the substrate comprises an amino acid sequence cleavable by the protease of the biosensor.

27. The composition or kit of claim 25 , further comprising an amplifier molecule.

28. The composition or kit of claim 25 , further comprising a deactivating molecule.

29. The composition or kit of claim 28 , further comprising an amplifier molecule operable with the biosensor, wherein the amplifier molecule comprises: (i) an amino acid sequence of a protease that is different to the protease(s) of the biosensor, (ii) an inhibitor of the protease of (i), and (iii) a linker amino acid sequence which comprises a cleavage site for the protease(s) of the biosensor; wherein the deactivating molecule comprises: (i) an amino acid sequence of a protease that is different to the protease(s) of the biosensor and that is different to the protease of the amplifier molecule; (ii) an inhibitor of the protease of (i) and (iii) a linker amino acid sequence which comprises a cleavage site for the protease of the amplifier molecule.

30. The composition or kit of claim 25 , further comprising an amplifier molecule operable with the biosensor, wherein the amplifier molecule comprises: (i) an amino acid sequence of a protease that is different to the protease(s) of the biosensor, (ii) an inhibitor of the protease of (i), and (iii) a linker amino acid sequence which comprises a cleavage site for the protease(s) of the biosensor; wherein the substrate comprises an amino acid sequence cleavable by the protease of the amplifier molecule.

31. The composition or kit of claim 25 , wherein the substrate comprises a label.

32. A method of detecting a target molecule, said method including the step of contacting the composition of claim 25 with a sample to thereby determine the presence or absence of a target molecule in the sample.

Assignees

• Univ Queensland

Inventors

• Alexandrov Kirill
• Stein Viktor
• Trau Matt

Classifications

• C07K2319/21

  containing a His-tag · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• G01N2333/18

  Togaviridae; Flaviviridae · CPC title

• C07K16/36

  against blood coagulation factors · CPC title

• C12N9/506

  derived from RNA viruses · CPC title