System for detecting protease

• US 7,109,293 B2
• Filed: 08/08/2002
• Issued: 09/19/2006
• Est. Priority Date: 08/10/2001
• Status: Expired due to Fees

• Current Assignee: Ahram Biosystems, Inc.
• Original Assignee: Ahram Biosystems, Inc.
• Inventors: Hwang, Inhwan, Lee, Yong Jik, Kim, Dae Heon
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US10/214,932
• Publication Number:
  US 20030100707A1
• Time in Patent Office: 1,503 Days
• Field of Search: 435/69.1, 530/350
• US Class Current: 530/350
• 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 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

Disclosed is a system for detecting a protease inside a cell. In one embodiment, the system includes a chimeric protein that comprises as covalently linked components: 1) at least one optionally masked signal protein; 2) at least one protease-specific cleavage site; and 3) at least one detectable amino acid sequence. The invention has a wide spectrum of applications including use in the detection of novel protease inhibitors inside cells and tissue.

Claims

1. A chimeric protein comprising at least one signal protein that has a trafficking signal targeting to a subcellular organelle and at least one proteolytic cleavage site for a protease, which is constructed such that

(a) the trafficking signals of all the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is present in cytosol;

(b) the trafficking signal of at least one signal protein is activated when the proteolytic cleavage site is cleaved by the protease, and as a result at least one fragment protein that includes the activated signal protein is transported to a subcellular organelle; and

(c) the chimeric protein is labeled with at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence signal in the cell is altered depending on the cleavage by the protease.

3. The chimeric protein according to claim 1 or 2, wherein among the fragment proteins produced by the proteolytic cleavage, at least two fragment proteins with different cellular localization characteristics includes different detectable amino acid sequences.

4. The chimeric protein according to claim 1 or 2, wherein among the fragment proteins including a signal protein whose inactivated trafficking signal is activated by the proteolytic cleavage, at least one fragment protein includes the detectable amino acid sequence.

5. The chimeric protein according to claim 1 or 2, wherein the trafficking signal of the inactivated signal protein is a signal targeting to a subcellular organelle selected from the group consisting of mitochondria, chloroplast, and peroxisome.

6. The chimeric protein according to claim 1 or 2, wherein the signal protein that is inactivated is a full length protein selected from the group consisting of Arabidopsis outer envelope membrane protein 7 (AtOEP7), Rubisco small subunit (RbcS), Chlorophyll a/b binding protein (Cab), Rubisco activase (RA), F1-ATPase, and Peroxisome-targeting motif (SKL), or a portion thereof that includes the trafficking signal.

10. The chimeric protein according to claim 1 or 2, wherein the signal masking protein is selected from the group consisting of amino acids, peptides, and proteins.

11. The chimeric protein according to claim 1 or 2 wherein the detectable amino acid sequence is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof.

2. A chimeric protein comprising at least two signal proteins that have trafficking signals targeting to subcellular organelles and at least one proteolytic cleavage site for a protease, which is constructed such that

(a) the trafficking signal of one signal protein remains active, and those of the rest of the signal proteins are inactivated by linking the proteolytic site or a signal masking protein through the proteolytic site to the N- or C-terminus of the signal proteins, and thus the chimeric protein is transported to a specific subcellular organelle targeted by the trafficking signal of the active signal protein;

(b) at least one proteolytic site and at least one inactivated signal protein are exposed to cytosol after the chimeric protein is transported to the subcellular organelle;

(c) the trafficking signal of the at least one inactivated signal protein exposed to cytosol is activated when the proteolytic cleavage site is cleaved by the protease, and as a result the fragment protein that includes the activated signal protein is transported to a subcellular organelle that is different from the subcellular organelle to which the chimeric protein was transported; and

(d) the chimeric protein is labeled with at least one detectable amino acid sequence and the position and intensity distribution of the detectable amino acid sequence in the cell is altered depending on the cleavage by the protease.

7. The chimeric protein according to claim 2, wherein the trafficking signal of the signal protein remaining active is a signal targeting to one selected from the group consisting of outer membranes of mitochondria, chloroplast, and nucleus, peroxisome membrane, and plasma membrane.

8. The chimeric protein according to claim 2, wherein the signal protein remaining active is a protein that binds specifically to a specific phospholipid.

9. The chimeric protein according to claim 2, wherein the signal protein remaining active is a full length protein selected from the group consisting of Arabidopsis outer envelope membrane protein 7 (AtOEP7), H⁺-ATPase, Pleckstrin homology domain (PH), and pleckstrin homology domain of FAPP (family A (phosphoinositide binding specific) member 3), or a portion thereof that includes the trafficking signal.