Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-23
2002-11-12
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069700, C435S320100, C435S069100, C435S029000, C435S471000, C435S476000, C536S023100, C536S023400
Reexamination Certificate
active
06479237
ABSTRACT:
The present invention relates to methods for detecting or identifying proteins involved in the formation of a protein complex, or in the inhibition of the formation of a protein complex.
Among the methods currently available for studying interactions between proteins, the two-hybrid system, and more recently the three-hybrid system, have been found to be particularly effective methods.
The two-hybrid system is based on the reconstitution of a transcription activator in yeast (Fields S. and Song O., 1989; Chien C.-T. et al., 1991). A first protein is cloned by fusion with the DNA binding domain, while a second protein is cloned by fusion with the transcription activation domain. When the two proteins of interest interact together, a transcription activator is formed which allows the transcription of reporter genes.
Again using the two-hybrid principle, by inserting a DNA sequence encoding a third partner into the vectors used, a three-hybrid system has been developed by Zhang and Lautar (Zhang J. et al., 1996).
However, the two-hybrid and three-hybrid systems currently available have the major drawback that it is necessary to carry out a test of interaction with a negative control, which corresponds to performing the following two separate experiments:
a first experiment in which the host cells are transformed with vectors containing the DNA sequence encoding the test protein, and the DNA sequences encoding the potential partner(s) of this test protein, and
a second experiment which serves as a negative control, in which the host cells are transformed with vectors not containing the DNA sequence encoding the test protein, but only those encoding the potential partner(s) of this test protein.
These two experiments are distinct since the host cells are transformed with different vectors depending on the experiment carried out.
Besides the fact that this need to carry out these two separate experiments does not allow rapid monitoring of the influence of the test protein on the other partner(s), the absence of growth of these negative controls often results from possible mutations or recombinations in one of the vectors, thus leading to erroneous results.
One of the aims of the present invention is, specifically, to provide new methods for studying interactions between proteins which do not need two separate experiments to be carried out and are consequently simpler and faster to perform and more reliable than the methods currently provided.
The invention relates to the use of at least one conditional promoter for carrying out a method for detecting one (or more) protein(s) (or polypeptides) interacting directly or indirectly with at least two other proteins (or polypeptides) during the formation of a protein complex or during the inhibition of the formation of a protein complex.
The expression “protein interacting directly with at least two other proteins during the formation of a protein complex” means:
any protein which combines with at least two other proteins to form with the latter a protein complex consisting of at least three proteins, or
any protein which catalyses the formation of a protein complex between at least two proteins (but without combining with these proteins to form a protein complex), in particular by means of a mechanism of conformational modification of one or more of the partners of the protein complex.
The expression “protein interacting directly with at least two other proteins during the inhibition of the formation of a protein complex” means:
any protein which inhibits the formation of the protein complex consisting of at least these two other abovementioned proteins, in particular by means of a mechanism of competition with one of the partners of the protein complex, or
any protein which catalyses the inhibition of the formation of the protein complex consisting of at least these two other proteins, in particular by means of a mechanism of conformational modification of one or more of the partners of the said protein complex.
Among the conditional promoters which can be used in the context of the present invention, mention may be made of:
the promoter Met25 (Kerjan P. et al., 1986), which can be regulated as a function of the methionine concentration, or
the promoters GAL1 or GAL10 (Johnston and Davis, 1984), which can be regulated as a function of the galactose concentration.
The invention also relates to any method for detecting a protein as defined above which interacts with at least two given proteins, during the formation of a protein complex or during the inhibition of the formation of a protein complex, characterized in that it comprises:
a step of transforming host cells whose genome contains one or more reporter genes with one or more vectors such that the genome of these host cells contains the DNA sequence encoding the test protein capable of being detected as well as the DNA sequences encoding at least two given proteins, the transcription of at least one of the DNA sequences being placed under the control of a conditional promoter,
a comparison of the effects of repression of the conditional promoter with the effects of activation of this conditional promoter on the possible transcription of the abovementioned reporter gene(s), it being possible to correlate the results of this comparison with the detection of an interaction or, on the contrary, the absence of an interaction between the test protein and the given proteins.
The method described above for detecting a protein interacting directly or indirectly with at least two given proteins preferably comprises:
a step of transforming host cells whose genome contains one or more reporter genes with one or more vectors such that the genome of these host cells contains:
on the one hand the DNA sequence encoding the said test protein capable of interacting with at least two given proteins, the transcription of this DNA sequence being placed under the control of a conditional promoter, and
on the other hand the DNA sequences encoding the said given proteins,
a comparison of the effects of repression of the conditional promoter with the effects of activation of this conditional promoter on the possible transcription of the abovementioned reporter gene(s), it being possible to correlate the results of this comparison with the detection of an interaction or, on the contrary, the absence of interaction between the said test protein and the said given proteins.
In the text hereinabove and hereinbelow, the expression “transforming host cells with a vector” means any method for inserting the content of DNA sequences of a vector, in particular a vector of the plasmid type, into the genome of the said cells; the resulting host cells are denoted hereinabove and hereinbelow by the expression “transformed host cells”. The expression “genome of a cell” means all the DNA contained in a cell, i.e. the chromosomal, mitochondrial and episomal DNA.
Thus, the method of the invention has the advantage of not comprising two separate experiments as described above, one of which serving as a negative control by parallel culturing of host cells transformed with vectors not containing any DNA sequence encoding the test protein, for the purpose of comparison with the step of culturing the host cells transformed with vectors containing all the DNA sequences encoding the proteins capable of interacting with each other.
The reason for this is that the host cells cultured in the method of the present invention, either as a negative control (or an internal control) or to demonstrate an interaction between the proteins, are transformed with the same vectors, thus avoiding the production of erroneous results, as indicated above in the context of the methods currently provided in this field.
Thus, one of the particularly advantageous aspects of the methods of the invention is that the control is intrinsic to the experiment, thereby making these methods particularly suitable for screening a cDNA library or a degenerate synthetic library of oligonucleotides which are liable to contain one (or more) DNA sequence(s) encoding one
Attar Ricardo
Camonis Jacques
Egly Jean-Marc
Romero-Portillo Francisco
Tirode Franck
Guzo David
Institut National de la Sante et de la Recherche Medicale (Inser
Leffers, Jr. Gerald G.
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