ENZYME SENSORS, METHODS FOR PREPARING AND USING SUCH SENSORS, AND METHODS OF DETECTING PROTEASE ACTIVITY

• US 20100055728A1
• Filed: 08/06/2007
• Published: 03/04/2010
• Est. Priority Date: 08/04/2006
• Status: Active Grant

• Current Assignee: Georgia State University Research Foundation, Inc.
• Original Assignee: Georgia State University Research Foundation, Inc.
• Inventors: Yang, Jenny J., Chen, Ning
• Granted Patent: US 8,481,272 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/9015 Ligases (6)
• CPC Class Codes: G01N 2333/95 Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 2333/976 Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Embodiments of the present disclosure provide for enzyme sensors, protease sensors, methods for producing and using the enzyme and protease sensors, methods of detecting and/or measuring protease activity, methods for characterizing protease cellular activity, fusion proteins, polynucleotides, and vectors corresponding to the enzyme and protease sensors, kits, and the like.

Claims

1. An enzyme sensor comprising:

a) a molecular recognition motif that binds an analyte; and

b) an optically-active fluorescent host protein in which the molecular recognition motif is operatively linked to or integrated therein, and

wherein the interaction of the analyte to the molecular recognition motif produces a detectable change.

2. The enzyme sensor of claim 1, wherein the optically-active fluorescent host protein is a fluorescent protein.

3. The enzyme sensor of claim 2, wherein the optically-active fluorescent host protein is an enhanced green fluorescent protein (EGFP).

4. The enzyme sensor of claim 1, wherein the analyte is an enzyme.

5. The enzyme sensor of claim 4, wherein the enzyme sensor senses a protease or a ligase.

6. The enzyme sensor of claim 5, wherein the protease is selected from:

trypsin-like proteases or their zymogens, chymotrypsin-like proteases or their zymogens, thrombin-like proteases or their zymogens, and caspase-like proteases or their zymogens.

7. The enzyme sensor of claim 6, wherein the trypsin-like protease is trypsin.

8. The enzyme sensor of claim 6, wherein chymotrypsin-like protease is chymotrypsin.

9. The enzyme sensor of claim 6, wherein thrombin-like protease is thrombin.

10. The enzyme sensor of claim 6, wherein caspase-like protease is selected from:

caspase-3, caspase5, caspase-6, caspase-7, caspase-8, caspase-9.

11. The enzyme sensor of claim 10, wherein the caspase is caspase-3.

12. The enzyme sensor of claim 6, wherein the zymogen is selected from:

trypsinogen, chymotrypsinogen, prothrombin and procaspases.

13. The enzyme sensor of claim 1, wherein the molecular recognition motif includes an analyte binding site.

14. The enzyme sensor of claim 13, wherein the analyte binding site is selected from a protease binding motif and a ligase binding motif.

17. The enzyme sensor of claim 14, wherein the analyte binding site is selected from:

a cleavage site, a binding site where the analyte binds to the protease sensor, and an enzyme reaction site.

18. The enzyme sensor of claim 1, wherein the molecular recognition motif includes an analyte binding site and a structural motif, wherein the structural motif is selected from a primary structure motif, a secondary structure motif, a tertiary structure motif, quaternary structure motif, a fragment or domain.

19. The enzyme sensor of claim 18, wherein the structural motif is selected from:

a loop, a loop-helix, a helix-loop, a helix-loop-helix motif, a beta-loop-helix, and a beta-loop-beta.

20. The enzyme sensor of claim 19, wherein the helix-loop-helix motif is an EF-hand motif.

21. The enzyme sensor of claim 1, wherein the molecular recognition motif is located in a region sensitive to the chromophore environment and has solvent accessibility.

22. The enzyme sensor of claim 21, wherein the sensitive region is disposed in the loop between beta sheet 8 and beta sheet 9 of a fluorescent protein.

23. The enzyme sensor of claim 22, wherein the sensitive region is disposed in the loop between amino acids 172 and 173 of enhanced green fluorescent protein (EGFP).

24. The enzyme sensor of claim 1, wherein optically-active fluorescent host protein in which the molecular recognition motif is operatively linked to or integrated therein has an amino acid sequence selected from:

SEQ ID NO;

1-118.

15. The enzyme sensor of claim 15, where the protease binding motif is specific for a protease selected from:

trypsin, chymotrypsin, thrombin, and caspases.

16. The enzyme sensor of claim 15, where the ligase binding motif is specific for ligase.

25-62. -62. (canceled)

63. A fusion protein, comprising an optically-active fluorescent host protein in which a molecular recognition motif is operatively linked to or integrated therein that has an amino acid sequence selected from:

SEQ ID NO;

1-118.

64. (canceled)

65. A kit for detecting protease activity comprising, a protease sensor, related agents that can facilitate the delivery of the protein to its desired destination, and directions, wherein the protease sensor includes:

a) a molecular recognition motif that binds an analyte; and

b) an optically-active fluorescent host protein in which the molecular recognition motif is operatively linked to or integrated therein, and

wherein the interaction of the analyte to the molecular recognition motif produces a detectable change.

66. The kit of claim 65, wherein the optically-active fluorescent host protein in which the molecular recognition motif is operatively linked to or integrated therein has an amino acid sequence selected from:

SEQ ID NO;

1-118.

67-75. -75. (canceled)