Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-12
2001-06-05
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06242183
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods for detecting protein interactions and isolating novel proteins.
SUMMARY OF THE INVENTION
In general, the invention features methods for detecting interactions among proteins.
Accordingly, in one aspect, the invention features a method of determining whether a first protein is capable of physically interacting with a second protein. The method includes (a) providing a host cell which contains (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene which expresses a first fusion protein, the first fusion protein comprising the first protein covalently bonded to a binding moiety which is capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene which expresses a second fusion protein, the second fusion protein including the second protein covalently bonded to a gene activating moiety and being conformationally-constrained; and (b) measuring expression of the reporter gene as a measure of an interaction between the first and said second proteins.
Preferably, the second protein is a short peptide of at least 6 amino acids in length and is less than or equal to 60 amino acids in length; includes a randomly generated or intentionally designed peptide sequence; or is conformationally-constrained as a result of covalent bonding to a conformation-constraining protein, e.g., thioredoxin or a thioredoxin-like molecule. Where the second protein is covalently bonded to a conformationally constraining protein the invention features a polypeptide wherein the second protein is embedded within the conformation-constraining protein to which it is covalently bonded. Where the conformation-constraining protein is thioredoxin, the invention also features a method which includes a second protein which is conformationally-constrained by disulfide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of the second protein.
In another aspect, the invention features a method of detecting an interacting protein in a population of proteins, comprising: (a) providing a host cell which contains (i) a reporter gene operably linked to a DNA-binding-protein recognition site; and (ii) a fusion gene which expresses a fusion protein, the fusion protein including a test protein covalently bonded to a binding moiety which is capable of specifically binding to the DNA-binding-protein recognition site; (b) introducing into the host cell a second fusion gene which expresses a second fusion protein, the second fusion protein including one of said population of proteins covalently bonded to a gene activating moiety and being conformationally-constrained; and (c) measuring expression of the reporter gene. Preferably, the population of proteins includes short peptides of between 1 and 60 amino acids in length.
The invention also features a method of detecting an interacting protein within a population wherein the population of proteins is a set of randomly generated or intentionally designed peptide sequences, or where the population of proteins is conformationally-constrained by covalently bonding to a conformation-constraining protein. Preferably, where the population of proteins is conformationally-constrained by covalent bonding to a conformation-constraining protein, the population of proteins is embedded within the conformation-constraining protein. The invention further features a method of detecting an interacting protein within a population wherein the conformation-constraining protein is thioredoxin. Preferably, the population of proteins is inserted into the active site loop of the thioredoxin.
The invention further features a method wherein each of the population of proteins is conformationally-constrained by disulfide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of said protein.
In preferred embodiments of various aspects, the host cell is yeast; the DNA binding domain is LexA; and/or the reporter gene is assayed by a color reaction or by cell viability.
In another related aspect, the invention features a method of identifying a candidate interactor. The method includes (a) providing a reporter gene operably linked to a DNA-binding-protein recognition site; (b) providing a first fusion protein, which includes a first protein covalently bonded to a binding moiety which is capable of specifically binding to the DNA-binding-protein recognition site; (c) providing a second fusion protein, which includes a second protein covalently bonded to a gene activating moiety and being conformationally-constrained, the second protein being capable of interacting with said first protein; (d) contacting said candidate interactor with said first protein and/or said second protein; and (e) measuring expression of said reporter gene.
The invention features a method of identifying a candidate interactor wherein the first fusion protein is provided by providing a first fusion gene which expresses the first fusion protein and wherein the second fusion protein is provided by providing a second fusion gene which expresses said second fusion protein. (Alternatively, the reporter gene, the first fusion gene, and the second fusion gene are included on a single piece of DNA.)
The invention also features a method of identifying candidate interactors wherein the first fusion protein and the second fusion protein are permitted to interact prior to contact with said candidate interactor, and a related method wherein the first fusion protein and the candidate interactor are permitted to interact prior to contact with said second fusion protein.
In a preferred embodiment, the candidate interactor is conformationally-constrained. Where the candidate interactor is an antagonist, reporter gene expression is reduced. Where the candidate interactor is an agonist, reporter gene expression i increased. The candidate interactor is a member selected from the group consisting of proteins, polynucleotides, and small molecules. In addition, a candidate interactor can be encoded by a member of a cDNA or synthetic DNA library. Moreover, the candidate interactor can be a mutated form of said first fusion protein or said second fusion protein.
In a related aspect, the invention features a population of eukaryotic cells, each cell having a recombinant DNA molecule encoding a conformationally-constrained intracellular peptide, there being at least 100 different recombinant molecules in the population, each molecule being in at least one cell of said population.
Preferably, the intracellular peptides within the population of cells are conformationally-constrained because they are covalently bonded to a conformation-constraining protein.
In preferred embodiments the intracellular peptide is embedded within the conformation-constraining protein, preferably thioredoxin; the intracellular peptide is conformationally-constrained by disulfide bonds between cysteine residues in the amino-terminus and in the carboxy-terminus of said second protein; the population of eukaryotic cells are yeast cells; the recombinant DNA molecule further encodes a gene activating moiety covalently bonded to said intracellular peptide; and/or the intracellular peptide physically interacts with a second recombinant protein inside said eukaryotic cells.
In another aspect, the invention features a method of assaying an interaction between a first protein and a second protein. The method includes: (a) providing a reporter gene operably linked to a DNA-binding-protein recognition site; (b) providing a first fusion protein including a first protein covalently bonded to a binding moiety which is capable of specifically binding to the DNA-binding-protein recognition site; (c) providing a second fusion protein including a second protein which is conformationally constrained and covalently bonded to a gene activating moiety; (d) combining the reporter gene, the first fusion protein, and the second fusion protein; and (e) measuring expression of the reporter gene.
The invention
Brent Roger
Jessen Timm H.
McCoy John M.
Clark & Elbing LLP
Schwartzman Robert A.
The General Hospital Corporation
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