Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-01
2001-12-25
Wortman, Donna C. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007320, C435S029000, C435S488000, C435S007600, C530S350000
Reexamination Certificate
active
06333154
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention concerns a method for selecting a molecule, and kit thereof, a method for screening a molecule, and kit thereof, and a signal amplification system comprising a bacterial multi-hybrid system.
The present invention relates to 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, 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 interaction between the said molecule of interest and the said target ligand, and wherein a signal amplification is generated.
The present invention also relates to a method of selecting a molecule of interest, which is capable of binding to a target ligand, wherein the interaction between the said molecule of interest and the said target ligand is detected with a signal amplification system according to the invention, by means of generating a signal amplification and triggering transcriptional activation.
The present invention also relates to a method of screening for a substance capable of stimulating or inhibiting the interaction between a target ligand and a molecule of interest, wherein respectively the stimulating or the inhibiting activity is detected with a signal amplification system according to the invention, by means of generating a signal amplification and respectively of triggering or of abolishing transcriptional activation, and wherein said signal amplification and said triggered or abolished transcriptional activation are compared with those obtained from an identical signal amplification system without any substance.
Most biological processes involve specific protein-protein interactions. General methodologies to identify interacting proteins or to study these interactions have been extensively developed. Among them, the yeast two-hybrid system currently represents the most powerful in vivo approach to screen for polypeptides that could bind to a given target protein. Originally developed by Fields and coworkers [Fields, S. & Song, O. (1989)
Nature
340, 245-6; Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991)
Proc. Natl. Acad. Sci. USA.
88, 9578-82. Two American U.S. Pat. No. 5,283,173 granted on Feb. 1, 1994 (Fields, S. & Song, O.) and U.S. Pat. No. 5,468,614 granted on Nov. 21, 1995 (Fields, S. & Song, O.) are also incorporated by reference], it utilizes hybrid genes to detect protein-protein interactions by means of direct activation of a reporter-gene expression (Allen, J. B., Walberg, M. W., Edwards, M. C. & Elledge, S. J. (1995)
Trends Biochem. Sci.
20, 511-6; Transy, C. & Legrain, P. (1995) Mol. Biol. Rep. 21, 119-27).
In essence, the two putative protein partners are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively. A productive interaction between the two proteins of interest will bring the transcriptional activation domain in the proximity of the DNA-binding domain and will trigger directly the transcription of an adjacent reporter gene (usually lacZ or a nutritional marker) giving a screenable phenotype. As there is evidence that the transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation, as reported by Keegan et al. (1986)
Science
231, 699-407 and Ma and Ptashne (1987)
Cell
48, 847-853.
Recently, Rossi et al. (Rossi, F., Charlton, C. A. & Blau, H. M. (1997)
Proc. Natl. Acad. Sci. USA.
94, 8405-8410) described a different approach, a mammalian “two-hybrid” system, which uses &bgr;-galactosidase complementation (Ullmann, A., Jacob, F. & Monod, J. (1968)
J. Mol. Biol.
32, 1-13) to monitor protein-protein interactions in intact eukaryotic cells.
Phage display (Smith, G. P. (1985)
Science
228, 1315-7; Scott, J. K. & Smith, G. P. (1990)
Science
249, 386-90) and double-tagging assay (Germino, F. J., Wang, Z. X. & Weissman, S. M. (1993)
Proc. Natl. Acad. Sci. USA.
90, 933-7) represent alternative approaches to screen complex libraries of proteins for direct interaction with a given ligand. However, these techniques do not allow an in vivo selection of the relevant clones.
Another approach is described in the International Patent Application No. WO 96/40987 (Schatz, P. J. et al.), which provides random peptide libraries and methods for generating and screening libraries to identify peptides that bind to receptor molecules of interest, including antibodies. The peptide library is constructed so that the DNA binding protein-random peptide fusion product can bind to the recombinant DNA expression vector that encodes the fusion product that contains the peptide of interest. The method of generating the peptide library comprises the steps of (a) constructing a recombinant DNA vector that encodes a DNA binding protein and contains binding sites for the DNA binding protein; (b) inserting into the coding sequence of the DNA binding protein in a multiplicity of vectors of step (a) coding sequences for random peptides such that the resulting vectors encode different fusion proteins, each of which is composed of the DNA binding protein and a random peptide; (c) transforming host cells with the vectors of steps (b); and (d) culturing the host cells transformed in step (c) under conditions suitable for expression of the fusion proteins. Typically, a random peptide library will contain at least 10
6
to 10
8
different members, although library sizes of 10
8
to 10
13
can be achieved.
A novel variety of approach is defined in the International Patent Application No. WO 96/29429 (Wickens, M. & Fields, S.) related to a hybrid system to detect protein-RNA interactions using the same method of achievement as recited in the two above-mentioned American patents. This hybrid system has a first hybrid protein comprising a DNA-binding domain and a first RNA-binding domain, a second hybrid protein comprising a transcriptional activation domain and a second RNA-binding domain, and a hybrid RNA. The interaction between both the first RNA-binding domain and the hybrid RNA and the second RNA-binding domain and the hybrid RNA causes the transcriptional activation domain to activate transcription of the detectable gene.
Bartel, P. L., Roecklein, J. A., SenGupta, D. & Fields, S. (1996)
Nat. Genet.
12, 72-77 extended the approach of the typical two-hybrid system consisting in a known protein that forms a part of a DNA-binding domain hybrid, assayed against a library of all possible proteins present as transcriptional activation domain hybrids, using the genome of the bacteriophage T7, such that a second library of all possible proteins is fused to the DNA-binding domain to be analyzed. This genome-wide approach to the two-hybrid searches has identified 25 interactions among the proteins of T7.
SUMMARY OF THE INVENTION
The aim of the present invention is to provide a novel bacterial multi-hybrid system, and more preferably a two-hybrid system, in which proteins of interest are genetically fused to two complementary fragments of a catalytic domain of an enzyme, which provides significant advantages over the prior art.
Thus, the present invention provides 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, wherein the first fragment is fused to a m
Karimova Gouzel
Ladant Daniel
Ullmann Agnes
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Wortman Donna C.
Zeman Robert A.
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