Bacterial multi-hybrid system and applications thereof
First Claim
1. A bacterial multi-hybrid signal amplification system comprising:
- (a) a first chimeric polypeptide comprising a first fragment of an enzyme chosen from adenylate cyclase and guanylate cyclase;
(b) a second chimeric polypeptide comprising a second fragment of an enzyme chosen from adenylate cyclase and guanylate cyclase, or a modulating substance capable of activating adenylate cyclase or guanylate cyclase, and (c) a signal molecule precursor, wherein the first fragment is fused to a molecule of interest, and the second fragment or the modulating substance is fused to a target ligand, and wherein the activity of the enzyme is restored by the in vivo interaction between the molecule of interest and the target ligand and wherein a signal molecule is generated by the restored enzyme activity; and
wherein the in vivo interaction occurs in a bacterial cell.
2 Assignments
0 Petitions
Accused Products
Abstract
A signal amplification system comprises a bacterial multi-hybrid system, and more preferably a two-hybrid system, of at least two chimeric polypeptides containing a first chimeric polypeptide corresponding to a first fragment of an enzyme and a second chimeric polypeptide corresponding to a second fragment of an enzyme or a modulating substance capable of activating said enzyme. The first fragment is fused to a molecule of interest and the second fragment or the modulating substance is fused to a target ligand. The activity of the enzyme is restored by the in vivo interaction between the molecule of interest and the target ligand. Signal amplification is generated and, for example, triggers transcriptional activation. The signal amplification system is useful in a method of selecting a molecule of interest, which is capable of binding to target ligand, wherein the interaction between the molecule of interest and the target ligand is detected with the signal amplification system as a kit therefor. A method of screening for a substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest is also provided.
-
Citations
21 Claims
-
1. A bacterial multi-hybrid signal amplification system comprising:
-
(a) a first chimeric polypeptide comprising a first fragment of an enzyme chosen from adenylate cyclase and guanylate cyclase;
(b) a second chimeric polypeptide comprising a second fragment of an enzyme chosen from adenylate cyclase and guanylate cyclase, or a modulating substance capable of activating adenylate cyclase or guanylate cyclase, and (c) a signal molecule precursor, wherein the first fragment is fused to a molecule of interest, and the second fragment or the modulating substance is fused to a target ligand, and wherein the activity of the enzyme is restored by the in vivo interaction between the molecule of interest and the target ligand and wherein a signal molecule is generated by the restored enzyme activity; and
wherein the in vivo interaction occurs in a bacterial cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
(a) a fragment corresponding to amino acids 1 to 224 of adenylate cyclase and a fragment corresponding to amino acids 225 to 399 of adenylate cyclase;
(b) a fragment corresponding to amino acids 1 to 224 of adenylate cyclase and a fragment corresponding to amino acids 225 to 384 of adenylate cyclase;
(c) a fragment corresponding to amino acids 1 to 137 of adenylate cyclase and a fragment corresponding to amino acids 138 to 400 of adenylate cyclase;
(d) a fragment corresponding to amino acids 1 to 317 of adenylate cyclase and a fragment corresponding to amino acids 318 to 400 of adenylate cyclase; and
(e) two fragments from eukaryotic adenylate cyclase.
-
-
6. The bacterial multi-hybrid signal amplification system according to claim 4 or 5, wherein the first fragment is a fragment corresponding to amino acids 1 to 224 of Bordetella pertussis adenylate cyclase and the second fragment is a fragment corresponding to amino acids 225 to 399 of Bordetella pertussis adenylate cyclase.
-
7. The bacterial multi-hybrid signal amplification system according to any one of the claims 1 or 3, wherein the modulating substance is a natural activator of the enzyme, or a fragment of said natural activator.
-
8. The bacterial multi-hybrid signal amplification system according to claim 7, wherein the natural activator is calmodulin, or a fragment thereof, and said first fragment is mutated compared to the wild type enzyme.
-
9. The bacterial multi-hybrid signal amplification system according to claim 8, wherein the first fragment is a mutated fragment of the catalytic domain of Bordetella adenylate cyclase.
-
10. The bacterial multi-hybrid signal amplification system of claim 3, wherein the catalytic domain comprises the first 400 amino acid residues of the adenylate cyclase.
-
11. A kit for selecting a molecule of interest, said kit comprising:
-
(a) a bacterial multi-hybrid signal amplification system according to claim 1;
(b) a bacterial strain or eukaryotic cell line deficient in endogenous adenylate cyclase; and
(c) a medium allowing the detection of the interaction selected from the group consisting of i) indicator or selective medium as minimal medium supplemented with lactose or maltose as a unique carbon source, ii) medium with antibiotics, iii, medium to visualize fluorescence, iv) conventional medium, and v) medium which allows the sorting by the presence of the phage receptor.
-
-
12. The kit for selecting a molecule of interest according to claim 11, wherein the bacterial strain is an E. coli strain.
-
13. A kit for selecting a molecule of interest, said kit comprising:
-
(a) a bacterial multi-hybrid signal amplification system according to claim 1, wherein the molecule of interest is a mutant molecule compared to a known wild type molecule;
(b) a bacterial multi-hybrid signal amplification system according to claim 1, wherein the molecule of interest is a known wild type molecule as a control;
(c) a bacterial strain or eukaryotic cell line deficient in endogenous adenylate cyclase;
(d) a medium allowing the detection of the interaction selected from the group consisting of i) indicator or selective medium as minimal medium supplemented with lactose or maltose as a unique carbon source, ii) medium with antibiotics, iii) medium to visualize fluorescence, iv) conventional medium, and v) medium which allows the sorting by the presence of the phage receptor; and
(e) means for detecting whether the signal amplification system with the mutant molecule is enhanced or inhibited with respect to the signal amplification system with wild type.
-
-
14. The kit for selecting a molecule of interest according to claim 13, wherein the bacterial strain is an E. coli strain.
-
15. A kit for screening for a substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest, said kit comprising:
-
(a) a bacterial multi-hybrid signal amplification system according to claim 1 with the substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest;
(b) a bacterial multi-hybrid signal amplification system according to claim 1 without any substance as the control;
(c) a bacterial strain or eukaryotic cell line deficient in endogenous adenylate cyclase; and
(d) a medium allowing the detection of the interaction selected from the group consisting of i) indicator plate or selective medium as minimal medium supplemented with lactose or maltose as a unique carbon source, ii) medium with antibiotics, iii) medium to visualize fluorescence, iv) conventional medium, and v) medium which allows the sorting by the presence of the phage receptor; and
(e) means for detecting whether the signal amplification system with the substance is enhanced or inhibited with respect to the signal amplification system without any substance.
-
-
16. The kit for selecting a molecule of interest according to claim 15, wherein the bacterial strain is an E. coli strain.
-
17. The bacterial multi-hybrid signal amplification system according to claim 1, further comprising:
a substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest.
-
18. The kit for selecting a molecule of interest according to claim 16, wherein the bacterial strain is an E. coli strain.
-
19. The bacterial multi-hybrid signal amplification system according to claim 1, wherein the first fragment comprises the catalytic domain of Bordetella adenylate cyclase.
-
20. The bacterial multi-hybrid signal amplification system according to claim 19, wherein the second chimeric polypeptide comprises a modulating substance capable of activating adenylate cyclase.
-
21. The bacterial multi-hybrid signal amplification system according to claim 20, wherein the modulating substance is calmodulin.
Specification