Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-22
2003-06-24
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S254210, C435S320100, C435S325000
Reexamination Certificate
active
06582927
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to molecular biology and more specifically to a method for identifying protein pairs involved in protein—protein interactions.
BACKGROUND OF THE INVENTION
The phrase “protein—protein interaction” refers to the ability of two protein molecules to bind to each other so as to form a complex. Such protein—protein interactions are involved in a large variety of biological processes including, for example, signal transduction pathways, enzyme-substrate interactions, viral adhesions and the formation of antibody-antigen complexes.
Many proteins are capable of interacting with a number of other proteins. Identifying and characterizing such interactions are highly important in understanding biological mechanisms, signal transduction pathways, etc. in characterizing the molecular basis of various diseases as disorders and in the is design of therapies.
A defect in a protein preventing it from participating in a protein—protein interaction can. as may be appreciated, have deleterious effects on a cell.
The ability to identify and characterize protein—protein interactions permits the identification of the defects in such interactions associated with a diseased state. The identification of such defects provides a target for potential therapies to cure or ameliorate the disease. In addition, the identification and characterization of protein—protein interactions provides a means to screen for drugs that alter the interaction. Such drugs can be useful, for example, to treat a disease caused, at least in part, by an aberrant protein—protein interaction.
Methods for assaying protein—protein interactions have been reviewed in Allen et al.,
Trends Biochem. Sci.,
20:511-516, 1995.
Proteins involved in a protein—protein interaction can be identified by detecting the presence of the protein complex in a cell or in a body fluid, and purifying the proteins forming the complex by biochemical methods. Such methods of isolation, however, are extremely tedious, particularly when the protein is expressed at low levels or if only a few cells express the protein. Immobilization of proteins on membrane filters has more recently lead to the development of filter based assays using proteins translated from cDNA molecules obtained, for example, from phage. However, the filter based assays, while being more sensitive, are also often very tedious.
A genetic method of identifying protein—protein interactions has also been developed (Fields et al,
Nature,
340:245-246, 1989). In this method, known as the “two hybrid assay”, one protein is fused to a DNA binding domain (typically from the Gal4 protein) while another protein is fused to a strong transcription activation domain. Binding of the two proteins inside a cell thus generates a functional transcription factor that is detected by a change in phenotype of the cell due to the expression of genes whose transcription is under the control of Gal4 DNA elements. The two hybrid system however, suffers from several limitations. First, protein pairs in which one of the proteins possesses transcriptional activity on its own, obviously cannot be analyzed. This includes bona fide transcription factors as well as proteins containing domains that fortuitously interact with the transcription machinery. Another limitation of the two hybrid system results from the toxicity of many proteins, for example certain homeodomain proteins and cell cycle regulators, when expressed in the nucleus. Furthermore, the two hybrid system produces false positive or false negative results when one of the proteins undergoes a conformational chance in the nucleus.
Another genetic method, the “Sos Recruitment System” (SRS) has also been described (Aronheim, A.,
Mol Cell. Biol.,
17:3094-3102, 1997). This method is based on the observation that localization of the protein hSos (the Ras guanyl nucleotide exchange factor) at the plasma membrane is essential for activating the Ras pathway and is therefore essential for viability. A yeast strain, such as cdc25-2, containing a temperature sensitive allele of Cdc25, (a yeast homologue of hSos) is thus viable only at the permissive temperature (24° C.). In the SRS system, a first protein (the bait protein) is fused to hSos while a second protein (the prey protein) is fused to a membrane localization domain. A protein—protein interaction between the bait and prey proteins localizes hSos at the plasma membrane. This complements the Cdc25 mutation which is detected as cell growth at the restrictive temperature (36° C.). However, the SRS also exhibits several limitations. First, about 20-30% of all bait proteins fused to hSos result in prey-independent complementation of Cdc25, a fact which yields a relatively high unspecific background signal (“noise”). Another limitation of the SRS system is that the effector part of the hSos is relatively large (150 Kda). This tends to complicate the fusion to hSos of both large bait proteins as well as short bait proteins.
Another problem of the SRS system is due to the fact that Ras encoded proteins are able to bypass the Cdc25 mutation because the yeast GTPase activating proteins (IRA genes) hydrolyze GTP bound to mammalian Ras proteins rather inefficiently thus leaving the Ras proteins in their active GTP-bound form.
SUMMARY OF THE INVENTION
The present invention makes use of the fact that in order for it to function. Ras needs to be localized at the plasma membrane. This localization normally occurs via the covalent attachment of a lipid moiety to cysteine 186 that anchors Ras at the membrane. Ras contains a consensus CAAX box located at the C-terminal end which undergoes famesylation and subsequently palmitoylation. A Ras lacking the famesylation box (CAAX) is non-functional since it cannot be localized at the membrane. The present invention thus makes use of cells with a Ras that is mutated such that it cannot be localized at the membrane, e.g. lacking the farnesylation box or having a mutation therein. These cells are “engineered” such that they express two fusion proteins, one fusion protein comprising a first protein (referred to herein at times as the “bait”) and a Ras protein which is mutated such that it cannot bind to the plasma membrane, and another fusion protein which comprises a second protein (referred to herein at times as the “prey”) and a membrane localization domain. If the bait binds the prey then the Ras fused to the prey becomes localized at the membrane and can thereby function.
FIG. 1
shows a schematic representation of the invention. In panel A, a cell incapable of expressing a functional Ras is made to express a Ras that cannot be localized at the membrane (and is thus non-functional) fused to a bait protein. A putative prey protein has been localized at the plasma membrane. A protein—protein interaction between the prey and bait proteins (panel B) localizes Ras at the plasma membrane. This produces a functional Ras that is detected as a phenotypic change in the cell.
This Ras Recruiting System (RRS) has several advantages over the SRS system:
1. The Ras protein is relatively small, thereby overcoming several of the technical limitations and practical problems posed by the large size of Sos as described above.
2. The RRS system exhibits substantially less false positive results, as compared to the SRS, with mammalian cDNA expression library screens and therefore represents a more efficient system for characterizing interacting proteins.
The invention thus provides a method for identifying a protein—protein interaction between a first protein and a second protein comprising the steps of:
(a) expressing in a cell which is incapable of activating a Ras protein;
(aa) a first nucleic acid sequence encoding a first fusion protein, said first fusion protein comprising a Ras protein mutated such that it cannot localize at the cell membrane and does not require an exchange factor fused to said first protein; and
(ab) a second nucleic acid sequence encoding a second fusion protein said second fusion protein, comprising said second prote
Katcheves Konstantina T
Ketter James
Rappaport Family Institute for Research in the Medical Sciences
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