Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-10
2001-01-09
Brusca, John S. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S068100, C435S069900, C435S070200, C435S004000, C436S501000
Reexamination Certificate
active
06171792
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to protein interaction detection systems.
Genetic analysis is a tool for understanding the protein networks that govern biological processes. The manipulations performed by geneticists (e.g., staging of temperature sensitive mutants, construction and analysis of double mutants, and generation and observation of F1 and F2 progeny) define relationships between genes. These abstract relationships between genes often reflect underlying biological realities. For example, the epistasis relation may suggest that one gene normally acts on another to cause a phenotype, the allelic specific suppression relation may suggest that two gene products physically interact (Hartman and Roth,
Adv. Genet.
17:1-105 (1973); Jarvik and Botstein,
Proc. Natl. Acad. Sci. USA
70:2046-50 (1973)), and the dependency relation may suggest that the action of one gene product precedes that o<another in time (Hereford and Hartwell,
J. Mol. Biol.
84:445-61 (1974)).
Information obtained from these genetic manipulations is typically of very high quality, but is often relatively difficult to acquire. The recent increase in the rate of identification of new coding sequences has renewed interest in global systematic methods to understand gene function. These methods include the “two hybrid” or “interaction trap” methods which have been developed to assay contact between a single bait and interacting proteins (Fields and Song,
Nature
340:245-6 (1989); Chien et al.,
Proc. Natl. Acad. Sci. USA
88:9578-82 (1991); Gyuris et al.,
Cell
75:791-803 (1993); Durfee et al.,
Genes Dev.
7:555-69 (1993); Estojak et al.,
Mol. Cell. Biol.
15:5820-9 (1995). Contact between two proteins in these systems defines a physical relationship that is frequently of biological significance.
SUMMARY OF THE INVENTION
In general, the invention features a method for detecting a protein—protein interaction, involving: (a) providing a host cell which contains (i) a first reporter gene operably linked to a DNA sequence that includes a first protein binding site; (ii) a second reporter gene operably linked to a DNA sequence that includes a second protein binding site; (iii) a first fusion gene which expresses a first fusion protein, the first fusion protein including a first protein covalently bonded to a binding moiety which is capable of specifically binding to the first protein binding site; (iv) a second fusion gene which expresses a second fusion protein, the second fusion protein including a second protein covalently bonded to a binding moiety which is capable of specifically binding to the second protein binding site; and (v) a third fusion gene which expresses a third fusion protein, the third fusion protein including a third protein covalently bonded to a gene activating moiety; (b) measuring expression output of the first reporter gene as a measure of the interaction between the first and the third proteins; (c) measuring expression output of the second reporter gene as a measure of the interaction between the second and the third proteins; and (d) interpreting the expression output results of step (b) and step (c), whereby (i) increased output of both of the first and the second reporter genes indicates that the third fusion protein interacts with both of the first and the second fusion proteins; (ii) increased output of the first reporter gene but not the second reporter gene indicates that the third fusion protein interacts with the first fusion protein but not the second fusion protein; (iii) increased output of the second reporter gene but not the first reporter gene indicates that the third fusion protein interacts with the second fusion protein but not the first fusion protein; and (iv) no change in output in either of the first or the second reporter genes indicates that the third fusion protein does not interact with either of the first or the second fusion genes.
In a preferred embodiment, the method further involves comparing the expression output results of step (b) and step (c) with the expression output result measured in either (a) a first comparison host cell which contains (i) a reporter gene operably linked to a DNA sequence including a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including the first protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the third protein covalently bonded to a gene activating moiety; or (b) a second comparison host cell which contains (i) a reporter gene operably linked to a DNA sequence including a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including the second protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the third protein covalently bonded to a gene activating moiety; or (c) both of the first and the second comparison host cells. In addition, any number of comparisons may be made between multiple “two-bait” cells or between a “two-bait” cell and multiple “one-bait” cells, or both.
In other preferred embodiments, at least one of the first or the second reporter genes may be reduced in expression level; one of the first or the second protein binding sites is a tetracycline operator; one of the first or the second reporter genes is URA3 or lacZ; one of the first or the second reporter genes produces a signal that is received and detected by a second cell; the host cell is a yeast cell or a mammalian cell; the first and the second reporter genes may be expressed simultaneously; and the first protein and the second proteins are allelic variants.
In a second aspect, the invention features a method for detecting a protein that mediates a change in the state of another protein, involving: (a) providing a host cell which contains (i) a reporter gene operably linked to a DNA sequence including a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including a first protein covalently bonded to a binding moiety which is capable of specifically binding to the protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including a second protein which is capable of interacting with the first protein and which is covalently bonded to a gene activating moiety, wherein at least one of the first or the second proteins may exhibit a change in state; (b) allowing the first and the second proteins to interact; (c) measuring expression of the reporter gene as a measure of the interaction between the first and the second proteins; (d) introducing into the cell a third gene expressing a third protein; (e) measuring expression of the reporter gene, a change in the reporter gene expression in the presence of the third protein being an indication that the third protein mediates a change in the state of the first or the second protein leading to an alteration in the ability of the first protein and the second protein to interact.
In a related aspect, the invention features an alternative method for detecting a protein that mediates a change in the state of another protein, involving: (a) providing a first host cell which contains (i) a reporter gene operably linked to a DNA sequence including a protein binding site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein including a first protein covalently bonded to a binding moiety which is capable of specifically binding to the first protein binding site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including a second protein which is capable of interacting with the first protein and which is covalently bonded to a
Brent Roger
Lok Walter L.
Mendelsohn Andrew R.
Xu C. Wilson
Brusca John S.
Clark & Elbing LLP
Siu Stephen
The General Hospital Corporation
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