Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-13
2002-11-19
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210, C435S007310, C435S069100, C435S254200
Reexamination Certificate
active
06482603
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for detecting interactions between two proteins as well as detecting the modulation of those interactions. The present invention is based upon the discovery of a new non-nuclear system to detect the interactions between two proteins and is particularly useful for the detection of the interaction between two or more proteins wherein one of the proteins is associated with the cell membrane. Related methods, compositions and kits can be used to detect or assay the interactions between essentially any two proteins that can be expressed in a cell.
BACKGROUND OF THE INVENTION
A fundamental area of inquiry in biology is the analysis of interactions between proteins. Proteins are complex macromolecules made up of covalently linked chains of amino acids. Each protein assumes a unique three dimensional shape determined principally by its sequence of amino acids. Many proteins consist of smaller units termed domains, which are continuous stretches of amino acids able to fold independently from the rest of the protein. Some of the important forms of proteins are enzymes, polypeptide hormones, receptors, nutrient transporters, structural components of the cell, hemoglobins, antibodies, nucleoproteins, and components of viruses.
Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Protein-protein interactions are involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport; and in the interaction of receptors on a cell with growth factors and hormones. Products of oncogenes can give rise to neoplastic transformation through protein-protein interactions. For example, some oncogenes encode protein kinases whose enzymatic activity on cellular target proteins leads to the cancerous state. Other examples of protein-protein interaction include when a virus infects a cell by recognizing a polypeptide receptor on the surface and when platelets aggregate during thrombosis.
Protein-protein interactions have been generally studied in the past using biochemical techniques such as cross-linking, co-immunoprecipitation and co-fractionation by chromatography. A disadvantage of these techniques is that interacting proteins often exist in very low abundance and are, therefore, difficult to detect. Another major disadvantage is that these biochemical techniques involve only the proteins, not the genes encoding them. When an interaction is detected using biochemical methods, the newly identified protein often must be painstakingly isolated and then sequenced to enable the gene encoding it to be obtained. Another disadvantage is that these methods do not immediately provide information about which domains of the interacting proteins are involved in the interaction.
To alleviate the problems associated with the biochemical characterization of protein-protein interactions, genetic systems have been invented that are capable of rapidly detecting which proteins interact with a known protein, determining which domains of the proteins interact, and providing the genes for the newly identified interacting proteins. One such system is the yeast two-hybrid system wherein two proteins are expressed in yeast: one protein of interest fused to a DNA-binding domain and the other protein of interest fused to a transcriptional activation domain (Fields et al. (1989)
Nature
340:245; Gyuris et al. (1993)
Cell
75:791; Harper et al. (1993)
Cell
75:805; Serrano et al. (1993)
Nature
366:704; and Hannon et al. (1993)
Genes & Dev.
7:2378). If the proteins interact, they activate transcription of a reporter gene that contains a binding site for the DNA-binding protein.
Although the development of genetic systems that utilize direct activation of a reporter gene, such as the yeast two-hybrid systems, has greatly facilitated the study of protein-protein interactions, many problems remain to be solved. For instance, the yeast two-hybrid systems rely on interactions between the two proteins in the nucleus of the cell. Accordingly, yeast two-hybrid systems are not useful for the study of integral membrane protein interactions and cannot be used to test cell membrane impermeate drugs. Furthermore, the study of protein-protein interactions wherein one of the proteins is itself a transcriptional activator often results in the transcription of the reporter gene without interaction between the two proteins under study. Lastly, the yeast two-hybrid systems require that both proteins under study be expressed as fusion proteins resulting in the possible loss of function.
SUMMARY OF THE INVENTION
The present inventors have discovered a genetic system to study protein-protein interactions that solves many of the problems associated with existing genetic systems. This system utilizes the receptor-G protein signaling system present in yeast and other eukaryotic cells to study protein-protein interactions, including the interactions wherein one protein is an integral membrane protein.
The present invention includes kits, vectors and methods for detecting one or more interactions between two proteins, comprising the steps of providing a cell with a first protein fused to a G protein gamma subunit and a second protein; and determining whether the interaction of the G protein gamma subunit with an effector molecule has been modulated, thereby determining whether the first and second proteins interact.
The present invention also includes kits, vectors and methods to identify an agent that modulates at least one interaction between two proteins, comprising the steps of exposing a cell to the agent, the cell comprising a first protein fused to a G protein gamma subunit and a second protein; and determining whether the interaction of the G protein gamma subunit with an effector molecule has been modulated.
The present invention further provides kits, vectors and methods to identify binding partners of a protein, comprising the steps of transforming or transfecting host cells with a library comprising a population of nucleic acid molecules which express a first protein fused to a G protein gamma subunit to produce a host cell population, said population of nucleic acids differing with respect to the first protein fused to a G protein gamma subunit; transforming the host cell population with a vector which expresses a second protein; culturing said host cell under conditions which express said first and second protein; and determining the activity of an effector molecule which is capable of interacting with the G protein gamma subunit. dr
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B
.
FIGS. 1A and 1B
are schematics comparing the traditional yeast two-hybrid methods of detecting protein-protein interactions to the methods of the invention. Proteins X and Y of
FIG. 1B
correspond to the first and second proteins of the claimed methods, respectively.
FIGS. 2A-2B
.
FIGS. 2A and 2B
are schematics of pRS314-GAL-rbSec1-H6STE18 and pRS316-ADH-syntaxin.
FIGS. 3A-3B
.
FIG. 3A
represents the growth inhibition results of an assay to detect the ability of Sec-1 and syntaxin to interact and prevent pheromone induced growth inhibition.
FIG. 3B
is a schematic of the elements used in each growth inhibition assay.
REFERENCES:
patent: 5482835 (1996-01-01), King et al.
patent: 5739029 (1998-04-01), King et al.
patent: 5876951 (1999-03-01), Fowlkes et al.
Garcia et al., A Rat Brain SEC1 Homologue Related to Rop and UNC18 Interacts with Syntaxin,Proceedings of the National Academy of Science USA, 91:2003-2007 (1994).
Song et al., Regulation of Membrane and Subunit Interactions by N-Myristoylation of a G Protein &agr; Subunit in Yeast,Journal of Biological Chemistry, 271:20273-20283 (1996).
Vidal et al., Genetic Characterization of a Mammalian Protein-Protein Interaction Domain by Using a Yeast Reverse T
Dohlman Henrik G.
Ehrhard Kathleen N.
Morgan Lewis & Bockius
Mosher Mary E.
Yale University
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