Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-04-24
2004-08-03
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C008S094240, C008S094240
Reexamination Certificate
active
06770446
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to novel cells which express heterologous fused proteins and methods of screening for compounds having peptide-binding activity, wherein the methods employ the novel cells of the invention.
BACKGROUND OF THE INVENTION
The specific binding of a pair of peptides to each other triggers a vast number of functions in a living cell. For example, the specific binding of a ligand to a surface receptor serves as the trigger for cellular responses to many external signals. In mammals, cells respond to a wide variety of circulating peptide hormones, often through single transmembrane domain receptors. It is certainly recognized that the cytokine receptor superfamily illustrates the diverse aspects of cellular function and physiological responses. Recent examinations of cytokine receptor function have revealed differing ligand-receptor protein stoichiometries including both 2-protein (ligand/receptor) (Cunningham et al., 1991; Staten et al., 1993). and 3-protein (ligand/receptor/receptor or ligand/receptor/transducer) interactions (Young, 1992; Taga and Kishimoto, 1992; Mui and Miyajima, 1994). The intricacies of such protein associations have been investigated using in vitro, often laborious, methods (Fuh et al., 1992; 1993; Davis et al., 1993) because easily manipulated genetic expression systems have been unavailable. The present invention is directed to novel, modified host systems that can be used for such investigations of protein-protein interactions. These novel systems are significantly less laborious than existing methods.
Recently reported systems in the art refer to a “2-hybrid” system as discussed by Fields and Song (1989) and also by Chein et al. (1991). The 2-hybrid system involves differential interactions between the separable DNA binding and activation domains of the yeast transcriptional activator Gal4. Heterologous proteins are expressed as hybrid proteins fused to either half of Gal4 (see FIG. 1; Fields and Song, 1989; Chein et al., 1991 for discussion of the 2-hybrid system). The positive interaction of the heterologous proteins brings the two halves of the Gal4 protein in close proximity, activating expression of a scorable reporter gene. Many yeast-based platforms link the desired effect of a gene or drug of interest to a change in yeast cell phenotype through the use of reporter genes. In yeast systems, reporter genes have commonly focused on auxotrophy genes for cell growth on selective media, or the LacZ gene for colorometric endpoint using assays that detect B-galactosidase activity. Here, we describe in-depth the applied utility of luciferase, for example, from
Renilla reniformis
or
Photinus pyralis,
as a reporter gene in yeast. Although luciferase has been used extensively as a reporter gene in mammalian cells, analogous use in yeast has not been reported. We have designed, validated and implemented luciferase reporter(s) for use in various yeast platforms, such as yeast two-hybrid for protein interaction studies. Yeast two-hybrid luciferase reporters provide increases in assay sensitivity, speed, ease, signal:noise ratios, and provide high quality quantitative data to yeast-based assays for a myriad of target identification and drug discovery applications. Use of the luciferase reporter gene in yeast provides substantial improvements to yeast-based assays as used for gene identification, protein-protein interaction identification and characterization, high throughput screening platforms, automation of these platforms, investigations of G-protein coupled receptors (GPCRs), regulation of GPCR signaling pathways, and ion channel studies.
Two-hybrid systems are useful for determining whether a first test peptide sequence has binding activity for a second known peptide sequence, wherein the affinity of the test peptide for the known peptide is unknown. Such a system has been used to analyze intracellular proteins such as transcription factors and kinase-target protein interactions (Yang et al., 1992; Durfee et al., 1993; Li et al., 1994).
The novel modified yeast cells of this invention, which use luciferase as a reporter gene, and the novel methods incorporating these cells provide a significant advance for the study and discovery of peptide mimics, including ligand mimics and receptor mimics. At this time, no one has developed an efficient and specific screening system to investigate these areas. By employing in a cell a peptide binding pair for which the binding affinity is known, the present invention permits the investigation of peptide binding pairs, such as a ligand and receptor, wherein the peptides bind via extracellular interactions. The present invention creates exponential advantages for the discovery of compounds which can interact as ligands for specific receptors or transducers. Potential ligands include, but are not limited to, mammalian hormones with the receptors being a cognate extracellular ligand-binding peptide. Furthermore, the present invention describes the use of cell systems which express multiple heterologous proteins, including the two heterologous fused proteins to establish the specific and reversible binding of the ligand and receptor. The specific interaction of the above-described binding is readily detected by a measurable change in cellular phenotype, for example, growth on selective medium.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to the novel modified host cells for the expression of heterologous fusion proteins. The novel modified host cells comprise:
a) a gene sequence encoding a heterologous fusion protein; said fusion protein comprising a first peptide of a peptide binding pair, or segment of said first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein;
b) a gene sequence encoding a heterologous fusion protein, said fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment of said second peptide, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a);
c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof.
d) optionally, a deletion or mutation in the chromosomal DNA of the yeast host cell for the transcriptional activation protein if present in the host cell.
The novel modified host cells of the present invention can be used to determine the interaction of a test sample with a selected peptide of a peptide binding pair. For example, the cell can be used to determine the interaction of a test sample with a selected ligand or receptor.
A second aspect of the present invention relates to novel modified cells and screening methods which indicate the interaction of a test sample with a selected peptide and receptor by a recognizable change in phenotype. The cell exhibits the change in phenotype only in the presence of a test compound having binding affinity for a peptide of the peptide binding pair, for example, binding affinity for a ligand or its receptor.
A third aspect of the present invention relates to novel cells and screening methods which permit determining to which peptide of a peptide binding pair a test sample binds.
A fourth aspect of the present invention relates to novel cells which express three or more heterologous components for the study of higher order multi-protein associations between three or more peptides, for example, such as the study of ligand dependent dimerization.
A fifth aspect of the present invention relates to novel yeast cells comprising a luciferase gene as a reporter gene and screening methods that indicate the interaction of a test sample with a peptide binding pair by a recognizable change in phenotype consequent to expression of the luciferase gene.
Defined Terms:
The term peptide binding pair refers to any pair of peptides having a known binding affinity for which the DNA sequence is known or can be deduced. The peptides of the peptide binding pair mu
Cao Jian
Young Kathleen H.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
McKelvey Terry
Vogel Nancy T.
Wyeth
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