Bacterial two-hybrid system for protein interaction screening
First Claim
1. A signal amplification system comprising a bacterial multi-hybrid system of at least two chimeric polypeptides containing:
- (a) a first chimeric polypeptide corresponding to a first fragment of an enzyme;
(b) a second chimeric polypeptide corresponding to a second fragment of an enzyme or a modulating substance capable of activating said enzyme, 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 said molecule of interest and the said target ligand and wherein a signal amplification is generated; and
wherein signal amplification is performed in an E. coli strain selected from the group consisting of strain BTH101 having C.N.C.M. Deposit Accession No. I-2309 and strain DHM1 having C.N.C.M. Deposit Accession No. 1-2310.
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Abstract
Two bacterial strains deficient in endogenous adenylate cyclase activity are provided. The strains, designated BTH101 and DHM1, are useful in a signal amplification system comprising 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 as described in published PCT application WO 99/28746. 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, and the kit therefor, as described in WO 99/28746. The strains are also useful in the method of screening for a substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest, and the kit therefor, as described in WO 99/28746.
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Citations
49 Claims
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1. A signal amplification system comprising a bacterial multi-hybrid system of at least two chimeric polypeptides containing:
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(a) a first chimeric polypeptide corresponding to a first fragment of an enzyme;
(b) a second chimeric polypeptide corresponding to a second fragment of an enzyme or a modulating substance capable of activating said enzyme, 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 said molecule of interest and the said target ligand and wherein a signal amplification is generated; and
wherein signal amplification is performed in an E. coli strain selected from the group consisting of strain BTH101 having C.N.C.M. Deposit Accession No. I-2309 and strain DHM1 having C.N.C.M. Deposit Accession No. 1-2310. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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- 10. A DNA library containing a collection of vectors transformed in a bacterial multi-hybrid system, wherein each vector contains a polynucleotide coding for the molecule of interest fused to a polynucleotide encoding for a first or second fragment of an enzyme.
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48. Strain BTH101 having C.N.C.M. Deposit Accession No. I-2309.
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49. Strain DHM1 having C.N.C.M. Deposit Accession No. I-2310.
Specification