Enzyme sensors, methods for preparing and using such sensors, and methods of detecting protease activity
View Patent ↗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.
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, 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: 39, 65-67, 71-78, 92-97, and 111-118, 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 analyte is an enzyme.
3. The enzyme sensor of claim 1 , wherein the molecular recognition motif includes an analyte binding site.
4. The enzyme sensor of claim 3 , wherein the analyte binding site is selected from a protease binding motif and a ligase binding motif.
5. The enzyme sensor of claim 4 , where the protease binding motif is specific for a protease selected from: trypsin, chymotrypsin, thrombin, and caspases.
6. The enzyme sensor of claim 4 , 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.
7. 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.
8. The enzyme sensor of claim 7 , 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.
9. The enzyme sensor of claim 8 , wherein the helix-loop-helix motif is an EF-hand motif.
10. 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.
11. 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: 39, 65-67, 71-78, 92-97, and 111-118.
12. 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 instructions for use of the kit, 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 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: 39, 65-67, 71-78, 92-97, and 111-118, and
wherein the interaction of the analyte to the molecular recognition motif produces a detectable change.