Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-05-06
2001-09-04
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254210, C435S254230
Reexamination Certificate
active
06284519
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 this 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) since cenetically malleable expression systems have been unavailable. The present invention is directed to novel modified host systems which can be used for such protein investigations, yet the novel systems are significantly less laborious.
Recently reported systems in the art refer to a “2-hybrid” system as discussed by Fields and Song, 1989 and also Chien et al., 1991. The “2-hybrid” sytem 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; Chien et al., 1991 for discussion of the 2-hybrid system). The productive interaction of the heterologous proteins brings the two halves of the Gal4 protein in close proximity, activating expression of a scorable reporter gene. To this date, such 2-hybrid systems have been disclosed as useful for determining whether a first given test peptide sequence has binding activity for the known sequence of a second peptide; wherein the affinity of the test peptide for the known peptide is unknown. Studies using such a system have been directed to analyzing 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 cells of this invention and novel methods incorporating these cells provide significant advancements 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 the 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 or 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, e.g. growth on selective medium.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to 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.
These 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; e.g. the cell can be used to determine the interaction of a test sample with 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 test compound having binding affinity for a peptide of the peptide binding pair, e.g. 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 (e.g. such as the study of ligand dependent dimerization).
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 must exhibit preferential binding for each other over any other components of the modified cell.
The term peptide as used in the above summary and herein means any peptide, polypeptide or protein, unless stated otherwise. As noted above, the peptides of a peptide binding pair can be a ligand and its corresponding receptor, or a ligand and any peptide having a known binding affinity for the ligand.
Heterologous as used in the above summary and herein means peptides which (1) are not expressed by the naturally-occurring host cell or (2) are expressed by the modified host cell by an expression method other than the expression method by which the host cell would normally express the peptide.
Unless specified otherwise, the term receptor as used herein encompasses the terms receptor
r
, soluble receptor, transducer and binding protein. In preferred embodiments of the invention, the receptor employed is a receptor, or soluble receptor, with receptor, being more preferred.
Receptor
r
as used herein means plasma membrane proteins that bind specific molecules, such as growth factors, hormones or neurotransmitters, and then transmits a signal to the cells' interior that causes a cell to respond in a specific manner. This includes single transmembrane proteins.
Soluble receptor means a non-transmembrane form of a recentor which is able to bind ligand. These are receozors released from a cell either by proteolysis or by alternatively spliced mRNA.
Binding protein means proteins that demonstrate binding affinity for a specific ligand.
Binding proteins may be produced from separate and distinct genes. For a given ligand, the binding proteins that are produced from specific genes are distinct from the ligand binding domain of the receptor or its soluble receptor.
Transducer means a
Ozenberger Bradley A.
Young Kathleen H.
American Cyanamid Company
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Schwartzman Robert A.
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