Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-07-22
2001-11-13
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100
Reexamination Certificate
active
06316223
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to compositions and methods for a genetic system of detecting protein-protein interactions in a mammalian host cell. The protein interactions are detected by using fusion proteins whose expression and interaction with each other results in transcriptional activation.
BACKGROUND OF THE INVENTION
Protein-protein interactions are of paramount and fundamental interest in biological systems. These interactions are involved in a wide variety of important biological reactions, including the assembly of enzyme subunits, in antigen-antibody reactions, in supramolecular structures of ribosomes, filaments, and viruses, in recognition and transport, in transcription regulation, and in ligand-receptor interactions. In addition, the area of protein-protein interactions has received significant attention in the area of signal transduction and biochemical pathway analysis.
Traditionally, protein-protein interactions were evaluated using biochemical techniques, including chemical cross-linking, co-immunoprecipitation and co-fractionation and -purification. Recently genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, and was termed the “yeast two-hybrid” system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463.
However, while the yeast system works well, it is unsuitable for use in mammalian systems for a variety of reasons. Furthermore, the existing mammalian two-hybrid systems are neither suitable for a wide variety of cells, nor flexible, as they generally require quite highly specialized conditions. In addition, the existing mammalian two-hybrid systems are generally transient systems, rather than stable systems. Finally, these systems tend to have high background signals from non-specific interactions, giving rise to “false positives”.
A number of factors make a flexible mammalian two-hybrid system highly desirable. First of all, post-translatonal modifications of proteins may contribute significantly to their ability to interact, yet mammalian cells have different post-translational modification systems than yeast. Thus, proteins that interact in a yeast system may not interact with the same specificity or avidity when placed in a mammalian cell. Similarly, proteins that would interact with correct post-translational processing may not be identified in a yeast system. In addition, a mammalian two-hybrid system that could be used in a wide variety of mammalian cell types would be highly desirable, since the regulation, induction, processing, etc. of specific proteins within a particular cell type can vary significantly; it would thus be a distinct advantage to assay for relevant protein-protein interactions in the relevant cell type. For example, proteins involved in a disease state could be tested in the relevant disease cells, resulting in a higher chance of identifying important protein interactions. Similarly, for testing of random proteins, assaying them under the relevant cellular conditions will give the highest chance of positive results. Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc., that may contribute to conditions which can effect protein-protein interactions. In particular, a mammalian protein interaction cloning system is useful to identify candidate bioactive agents that have the potential to modulate a given protein-protein interaction.
Thus, a robust and adaptable mammalian two-hybrid system which can work in a wide variety of mammalian cell types is highly desirable.
Accordingly, it is an object of the invention to provide compositions and methods useful in a two-hybrid system which can be utilized reproducibly and stably in mammalian cells.
SUMMARY OF THE INVENTION
The invention provides compositions and methods useful in a mammalian two hybrid system for the detection of protein-protein interactions.
In one aspect of the invention, the invention provides compositions comprising a bait vector and a test vector. The bait vector comprises a first selection gene, a bait vector viral origin of replication which requires a bound viral replication protein to effect replication, and a first fusion gene. The fusion gene comprises a first sequence encoding a nucleic acid binding domain, and a second sequence comprising either a sequence encoding a bait protein or a cloning sequence comprising at least one cloning site. The test vector comprises a second selection gene, a test vector viral origin of replication which requires a bound viral replication protein to effect replication, and a second fusion gene. The second fusion gene comprises a third sequence encoding a transcriptional activation domain, and a fourth sequence comprising either a sequence encoding a test protein or a cloning sequence comprising at least one cloning site.
In a further aspect, the compositions further comprise a reporter vector comprising a first detectable gene, a viral replication protein gene, and an operator site, which upon binding of the nucleic acid binding domain and the transcriptional activation domain due to a protein-protein interaction of the bait protein and the test protein, will activate transcription of the first detectable gene and the viral replication protein gene.
In a further aspect, the invention provides compositions comprising a retroviral bait vector comprising a first fusion gene and an optional selection gene, and a retroviral test vector comprising a second fusion gene and an optional selection gene. The composition may further comprise a retroviral reporter vector comprising a first detectable gene and an operator site.
In an additional aspect, the invention provides mammalian host cells containing the compositions of the invention, optionally stably integrated into their chromosomes.
In a further aspect, the invention provides methods for detecting an interaction between a bait protein and a test protein comprising providing a mammalian host cell comprising a bait vector, a test vector, and a reporter vector, which may or may not be retroviral vectors. The host cell is subjected to conditions under which the first fusion gene and the second fusion gene are expressed to produce a first fusion protein and a second fusion protein. The method further comprises determining whether a protein-protein interaction between the first fusion protein and the second fusion protein occurred.
In a further aspect, the invention provides methods for isolating the test protein. The sequence encoding the test protein is isolated. Sequences encoding the full-length protein are also isolated.
In a further aspect, the invention provides methods for screening for bioactive agents that have the potential to modulate the protein-protein interaction between a bait protein and a test protein. These methods comprise (1) providing a mammalian host cell comprising a vector composition comprising genes encoding a bait protein, a test protein and a reporter protein, (2) subjecting the host cell to conditions under which the genes are expressed, producing a bait protein, a test protein and a reporter protein, (3) determining whether a protein-protein interaction between the bait protein and the test protein occurred by determining expression of the reporter protein, (4) adding candidate bioactive agents that are capable of modulating this protein-protein interaction, (5) determining the effect of the candidate bioactive agents on the protein-protein interaction between the bait protein and
Huang Betty
Luo Ying
Payan Donald
Flehr Hohbach Test Albritton & Herbert LLP
Park Hankyel T.
Rigel Pharmaceuticals Inc.
Silva Robin M.
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