RAS-BINDING PROTEIN (PRE1)

Abstract: Disclosed is a Ras-binding protein designated PRE1. PRE1 occurs naturally in various mammalian tissues and cell types. An isolated DNA endocing PRE1, vectors and cells containing the DNA, and PRE1-specific antibodies are also disclosed. Also disclosed is an in vitro screening method for indentifying a substance which modulates PRE1-Ras binding, and an in vitro method for identifying a substance that modulates PRE1 gene expression.

Description: Ras-Binding Protein (PREl) Field of the Invention The invention relates to recombinant DNA, cell biology and oncology. Background of the Invention In humans, mutations in the cellular Ras gene (c-ras) which render Ras constitutively active, have been associated with different types of cancers (Bos et al . , Cancer Res . 94:4682-4689). Ras relays signals from receptor tyrosine kinases, (Fantl et al . , 1993, Annu . Rev. Biochem . , 62 :453-481) , non-tyrosine kinase receptors ( oodrow et al . , 1993, J. Immunol . , 150: 3853-3861) and heterotrimeric G protein-coupled receptors (Van Corven et al . , 1993, Proc . Nat ' l . Acad . Sci . , 90:1257-1261). Ras is located at the inner surface of the plasma membrane. Activation of cell surface receptors promotes the exchange of Ras-bound GDP for GTP, thereby causing a conformational change in Ras. This conformational change activates Ras so that it interacts with downstream targets or effectors ( ittinghofer et al . , 1996, Trends In Biochem . Sci . , 21:488-491. Several candidate Ras effectors have been proposed based on their ability to bind to Ras through its effector loop. Among these are: Raf , PI -3 kinase, members of the Ral-GDS family, Rin-1, AF-6, diacylglycerol kinases, PKC ζ , and MEKKl (See, e . g. , Katz et al. , 1997, Curr. Opin . Genet . Dev. , 7:75-79; Marshall, 1996, Curr. Opin . Cell Biol . , 8:197-204). Activation of effectors such as these leads to activation of other downstream signal transduction molecules. This signaling cascade culminates in the modulation of gene expression, and thereby causes changes in cellular function, growth, and division. Summary of the Invention A Ras effector protein, designated PREl, has been identified and characterized. PREl protein is expressed in various tissues and cell lines, and PREl binds to Ras. The invention features an isolated DNA containing a nucleotide sequence that encodes a PREl protein. The PREl protein encoded by the DNA shares at least 80% sequence identity with SEQ ID NO: 2, and it binds to Ras. The nucleotide sequence can define a DNA molecule whose complement hybridizes under high stringency conditions to a DNA whose nucleotide consists of SEQ ID NO:l. Preferably, the isolated DNA encodes a naturally-occuring mammalian PREl. In some embodiments, the DNA encodes an amino acid sequence consisting of SEQ ID NO:2. The DNA can contain the nucleotide sequence of SEQ ID NO:l or the coding region of the nucleotide sequence of SEQ ID NO:l, or degenerate variants of those sequences. The invention also includes a vector containing the above-described DNA, which DNA can be operably linked to one or more expression control sequences. The invention also includes a cell containing such a vector. The invention features a substantially pure PREl protein that includes an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 2 and binds to Ras. Preferably, the sequence identity is at least 85%, more preferably, it is at least 90%, and most preferably, it is at least 95%. The amino acid sequence of the PREl protein can differ from SEQ ID NO: 2 solely by conservative amino acid substitutions (i.e., substitution of one amino for another of the same class) or by non- conservative sustitutions, deletions, or insertions located at positions that do not destroy the function of the protein. In some embodiments, the protein has an amino acid sequence consisting of SEQ ID NO:2. Also included in the invention is any naturally-occurring homolog or isoform of SEQ ID N0:2. The invention includes Ras-binding domain-containing PREl protein fragments, e.g., amino acids 266-360 or 188-413, and heterologous fusion proteins containing a Ras-binding domain-containing PREl protein fragment. The invention also features a PREl-specific antibody, which can be polyclonal or monoclonal. The antibody can be conjugated to a detectable label. The invention also features a screening method for identifying a substance that modulates binding of PREl protein to Ras. The method includes the following steps: (a) providing a sample solution of PREl protein; (b) adding to the sample solution a candidate substance; (c) adding to the sample solution a Ras sample; and (d) detecting an increase or decrease in binding of PREl protein to Ras in the presence of the candidate substance, compared to the binding of PREl to Ras in the absence of the candidate substance . The invention also features a method of producing PREl protein. The method includes the following steps: (a) providing a cell transformed with an isolated DNA comprising a nucleotide sequence that encodes a protein the amino acid sequence of which is SEQ ID NO : 2 ; (b) culturing the cell; and (c) collecting the protein encoded by the nucleotide sequence. The invention also features a screening method for identifying a substance that modulates PREl gene expression. The method includes the following steps: (a) providing a test cell; (b) contacting the test cell with a candidate substance; and (c) detecting an increase or decrease in the level of PREl gene expression in the presence of the candidate substance, compared to the level of PREl gene expression in the absence of the candidate substance . The invention also features a method for isolating a PREl-binding substance, e.g., Ras protein. The method includes the following steps: (a) providing a sample of immobilized PREl protein; (b) contacting a mixture containing the substance with said immobilized PREl protein; (c) separating unbound components of the mixture from bound components of the mixture; (d) recovering the PREl-binding substance from said immobilized PREl protein. As used herein, "high stringency conditions" means the following: hybridization at 42 °C in the presence of 50% formamide; a first wash at 65°C with about 2 x SSC containing 1% SDS ; followed by a second wash at 65 °C with 0.1 x SSC. As used herein, "isolated DNA" means DNA free of the genes that flank the gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote . It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR) , or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Also included is a recombinant DNA that includes a portion of SEQ ID NO:l and that encodes an alternative splice variant of PREl. As used herein, "operably linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a gene of interest . As used herein, "protein" means any peptide-linked chain of amino acids, regardless of length or post- translational modification, e.g., glycosylation or phosphorylation. As used herein, "PREl" means: (1) a protein, the amino acid sequence of which is SEQ ID N0:2, or (2) a protein that shares at least 80% amino acid sequence identity with SEQ ID NO: 2 and binds to Ras. As used herein, "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10- nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . As used herein, "PREl-specific antibody" means an antibody that binds to a protein, the amino acid sequence of which is SEQ ID NO: 2 and displays no substantial binding to other naturally-occuring proteins other than those sharing the same antigenic determinants as PREl. The term includes polyclonal and monoclonal antibodies. As used herein, "substantially pure protein" means a protein separated from components that naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure PREl protein can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding an PREl polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically- synthesized protein or a recombinant protein produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure proteins include those having sequences derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes . As used herein, "fragment", as applied to a protein, means at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering. As used herein, "test cell" means a cell that expresses a PREl gene in the absence of a PREl gene repressor. Preferably, the PREl gene in the test cell is under the control of a promoter that is naturally associated with a PREl gene. As used herein, "vector" means a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will control. Unless otherwise indicated, materials, methods, and examples described herein are illustrative only and not intended to be limiting. Various features and advantages of the invention will be apparent from the following detailed description and from the claims. Brief Description of the Drawing Fig. 1 is the nucleotide sequence of a murine PREl cDNA, and the deduced amino acid sequence. Detailed Description PREl Structure and Function A full-length murine PREl cDNA has been cloned and sequenced. The PREl cDNA clone contains 3018 bp (Fig. 1) and includes a complete open reading frame that encodes a protein 413 amino acids in length. The sequence of the 3018 bp cDNA is shown in Fig. 1. The cDNA sequence around the first ATG matches the Kozak consensus sequence for a translational start site. The open reading frame from this methionine includes 413 amino acids, yielding a highly basic polypeptide (PI=9.41) with a predicted molecular weight of 46.4 KD. One structural feature of PREl is the presence of a cysteine-histidine rich segment (a. a. 118-165, H-X 13 -C-X 2 -C-X 10 -C-X 2 -C-X 4 -H-X 2 -C-X 7 -C) typical of a diacylglycerol/phorbol ester (DAG_PE) binding site (Ono et al.,1989, Proc .Natl .Acad . Sci , 86:4868-4871). PREl's carboxyterminal region shares some homology to the putative Ras-binding domains of other proteins (Ponting et al . , 1996, Trends In Biochem. Sci . 21:422-425) . PREl also has a proline rich region in its aminoterminal region, with five PXXP sequences (aa 17-20 PEPP; 31-34 PPPP; 34-37 PARP; 77-80 PVRP and 105-108 PQDP) , which are possible SH3 domain binding sites (Ren et al., 1993, Science 259:1157-1161). Ras interacts with downstream effectors to cause changes in cellular function, growth, and division. Mutations in this signaling pathway can lead to abnormal cell proliferation or neoplasia. Substances that block the Ras-effector interaction can thus inhibit abnormal cell proliferation or neoplasia. Screening for substances that modulate Ras-PREl interaction is therefore useful for identifying potential cancer therapy agents . PREl may be a catalytic signaling molecule. Preliminary experiments, however, indicate that prokaryotic recombinant PREl (a. a. 188-413) does not alter Ras-GTP exchange or Ras-GTPase activity in vi tro . PREl may also be an adapter molecule, serving to link activated Ras with another, presumably catalytic, signaling molecule. The Ras binding domain (a. a. 266- 360) in PREl is located in its carboxyterminal region, and there are potential SH3 binding sites in the aminoterminal region. PREl may link a SH3 domain- containing protein to activated Ras. PREl also contains a DAG_PE binding consensus sequence motif also found in a variety of proteins involved in intracellular signaling including Raf (Rapp et al . , 1988, Cold Spring Harb . Symp . Quant . Biol . , 53 Pt.l : 173-184) , PKC (Ono et al.,1989, Proc . Natl .Acad . Sci . , 86:4868-4871), VAV (Ahmed et al . , 1992, Biochem. J. , 287:995-999), n-chimerin (Ahmed et al., 1990, Biochem. J. , 272:767-773) and DAG kinases (Sakane et al . , 1990, Nature, 344:345-348). DAG may bind to the DAG_PE site in PREl and regulate its function. Expression Control Sequences and Vectors The PREl protein-encoding DΝA ("PREl DΝA" ) of this invention can be used in a screening method to identify a substance that inhibits cell proliferation or neoplasia in a mammal. For such uses, the PREl DΝA is typically cloned into an expression vector, i.e., a vector wherein PREl DΝA is operably linked to expression control sequences. The need for, and identity of, expression control sequences will vary according to the type of cell in which the PREl DΝA is to be expressed. Generally, expression control sequences include a transcriptional promoter, enhancer, suitable mRΝA ribosomal binding sites, and sequences that terminate transcription and translation. Suitable expression control sequences can be selected by one of ordinary skill in the art. Standard methods can be used by the skilled person to construct expression vectors. See generally, Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manual (2nd Edition) , Cold Spring Harbor Press, Cold Spring Harbor, ΝY. Vectors useful in this invention include plasmid vectors and viral vectors. Preferred viral vectors are those derived from retroviruses, adenovirus, adeno- associated virus, SV40 virus, or herpes viruses. PREl DNA In Vi tro In some embodiments of the invention, PREl DNA is introduced into, and expressed in, a prokaryotic cell, e.g., Escherichia coli . For expression in a prokaryotic cell, PREl DNA can be integrated into a bacterial chromosome or expressed from an extrachromosomal DNA. In other embodiments of the invention, the PREl DNA is introduced into, and expressed in, a eukaryotic cell in vi tro . Eukaryotic cells useful for expressing PREl DNA in vi tro include, but are not limited to, COS, CHO, and Sf9 cells. Transfection of the eukaryotic cell can be transient or stable. The PREl DNA can be, but is not necessarily, integrated into a chromosome of the eukaryotic cell. PREl-Specific Antibody Some embodiments of this invention include a PRE1- specific antibody. Standard protocols for monoclonal and polyclonal antibody production are known and can be carried out by one of ordinary skill in the art to obtain antibodies useful in this invention. The invention encompasses not only an intact monoclonal or polyclonal antibody, but also an immunologically active antibody fragment. Examples of such a fragment include a Fab or F(ab 2 ) fragment, an engineered single chain Fv molecule, and a chimeric antibody. Typically, a chimeric antibody includes a variable region of a non-human antibody, e.g., a murine variable region, and a constant region of a human antibody. Antibody Label In some embodiments of the invention, the PRE1- specific antibody includes a detectable label. Various types of detectable labels can be linked to, or incorporated into, an antibody of this invention. Examples of useful label types include radioactive, non- radioactive isotopic, fluorescent, chemiluminescent, paramagnetic, enzyme, or colorimetric . Examples of useful enzyme labels include malate hydrogenase, staphylococcal dehydrogenase, delta-5- steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, and glucoamylase, acetylcholinesteras . Examples of useful radioisotopic labels include 3 H, 131 I, 125 I, 32 P, 35 S, and 14 C. Examples of useful fluorescent labels include fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorescamine . Examples of useful chemiluminescent label types include luminal, isoluminal, aromatic acridinium ester, imidazole, acridinium salt, oxalate ester, luciferin, luciferase, and aequorin. Suitable labels can be coupled to, or incorporated into antibodies or antibody fragments through standard techniques known to those of ordinary skill in the art. Typical techniques are described by Kennedy et al . , (1976) Clin . Chim . Acta 70, 1-31; and Schurs et al . , (1977) Clin . Chim. Acta 81, 1-40. Useful chemical coupling methods include those that use glutaraldehyde, periodate, dimaleimide and m-maleimido-benzyl-N-hydroxy- succinimide ester. Screening assays The invention can be used to screen candidate substances for the ability to inhibit the interaction of Ras with PREl . In an exemplary screening method, the two-hybrid expression system described below is used to screen for substances capable of inhibiting Ras-PREl interaction in vivo . The two-hybrid method is a well known yeast-based genetic assay to detect protein-protein interactions in vivo ( See, e . g. , Bartel et al . , 1993, In Cellular Interactions in Development : A Practical Approach, Oxford University Press, Oxford, pp. 153-179; Chien et al . , 1991, Proc . Natl . Acad . Sci . USA, 88:9578-9582; Fields et al., 1989, Nature, 340:245-247; Fritz et al . , 1992, Curr. Biol . , 2:403-405; Guarente, L., 1993, Proc . Natl . Acad. Sci . USA, 90:1639-1641). In this system, a GAL4 binding site, linked to a reporter gene such as lacZ, is contacted in the presence and absence of a candidate substance with a GAL4 binding domain linked to a PREl fragment and a GAL4 transactivation domain II linked to a Ras fragment. Expression of the reporter gene is monitored, and a decrease in its expression is an indication that the candidate substance inhibits the interaction of Ras with PREl. One of ordinary skill in the art will recognize that other screening assays are known and can be used to identify candidate substances that inhibit Ras-PREl interaction. In another screening method, one of the protein components of the Ras-PREl binding complex, such as Ras or a PREl-binding fragment of Ras or PRE-1 or a Ras- binding fragment of PRE-1, is immobilized. Polypeptides can be immobilized using methods known in the art. Such methods include adsorption onto a plastic microtiter plate or specific binding of a glutathione-S-transferase (GST) -fusion protein to a polymeric bead containing glutathione. For example, GST-PRE1 (a. a. 188-413) can be bound to glutathione-Sepharose™ beads. The immobilized protein (e.g., PREl) is then contacted with the labeled protein to which it binds (Ras in this example) in the presence and absence of a candidate substance . Unbound protein can be removed by washing. The complex then can be solubilized and analyzed to determine the amount of bound (labeled) protein. A decrease in binding is an indication that the candidate substance inhibits the interaction of Ras and PREl. A variation of the above-described screening method can be used to screen for other classes of candidate substances, e . g. , those that disrupt previously-formed Ras-PREl complexes. In this variation, a complex containing Ras (or a PREl-binding Ras fragment) bound to PREl (or a Ras-binding fragment) is immobilized and contacted with a candidate compound. Detection of disruption of the Ras-PREl complex by the candidate substance identifies the candidate substance as a potential modulator of Ras-mediated cellular events. The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way. Examples Cloning of PREl cDNA A yeast two hybrid screen was carried out to identify potential new Ras effectors in mammalian cells. A cDNA encoding V12-H-Ras without the last four amino acids was subcloned into vector pAS-CYH-II carboxyterminal to the Gal -4 DNA binding domain. This formed the bait construct pAS-Ras, which was used to transform the yeast strain Y190. Stable transformants were selected on Trp(-) plates and a single yeast colony was picked, grown up in Trp medium and saved. After verifying that V12-H-Ras was correctly expressed in this yeast clone, 100 μg of cDNA made from an activated mouse T cell library constructed in the GAL- 4 DNA activation domain vector pACT was transformed into one million yeast cells expressing pAS-Ras. The transformants were plated out on 10 large His(-)Leu(- )Trp(-) selection plates. After eight days, 20 large colonies appeared. An X-gal filter assay was performed for all the colonies and all showed strong blue color, indicative of lacZ activity. The colonies were individually picked. Yeast plasmid DNA was isolated from each picked colony and transformed into bacterial strain WA921 by electroporation. Transformants were subjected to selection on on Leu(-) LB plus ampicillin plates. The bait plasmid pAS-Ras was usually removed by this selection, and the pACT-cDNA plasmids were isolated from the bacteria using conventional methods. Of the positive clones, two encoded a 2.5 Kb cDNA representing a new gene, which was designated PREl. The 2.5 Kb cDNA encoding PREl from the initial two-hybrid screen was isolated, labeled with [cκ- 32 P] dCTP and used to screen cDNA libraries from mouse brain (Clontech's mouse brain 5' -stretch plus cDNA library in λ-gt 10 vector, CAT. #ML 3000a) . A positive clone containing a 3018 bp insert was isolated. By low stringency screening of cDNA libraries, at least two more cDNAs encoding polypeptides with regions similar or identical to regions of PREl were obtained. They diverged substantially from PREl in sequences aminoterminal to the Cys-His rich segment. These may represent alternative splice variants of PREl gene products or genes related to PREl . PREl-Specific Antibody A polyclonal antiserum was raised against a carboxyterminal fragment of PREl (a. a. 188-413) using conventional methods. A polyclonal antibody preparation was produced by affinity chromatography using the recombinant antigen. AGST-PRE1 (a. a. 188-213) fusion protein was used to immunize NZW rabbits. The antiserum was first depleted of GST-reacting antibodies by repeated incubation with immobilized GST. The GST depleted antiserum was then affinity purified using immobilized PREl (a. a. 188-413) on PVDF membrane. Northern blot PREl mRNA abundance and complexity in murine tissues was examined by Northern blot. A 210 bp PREl cDNA fragment (nt . 90-310) was labelled with 32 P-dCTP by the random priming method and used for probing PREl mRNA. The mouse multiple tissue blot was purchased from Clontech and hybridized in Stratagene's quick hybridization buffer and washed according to the manufacturer's protocol. A single mRNA containing about 3.1 Kb was detected in most mouse tissues, including: heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. Some mRNA size variation was noted. The highest levels were observed in brain, liver and spleen, with barely detectable levels in heart. Tissue and cell line western blot Western blots using polyclonal anti-PREl (a. a. 188-413) antibody were performed. Sprague-Dawley rats (65g) were starved overnight and anesthetized with pentobarbital . Tissues from the rats were excised in the following order: gastrocnemius, testis, spleen, kidney, liver, lung and heart. Brain was excised from other intact anesthetized animals after decapitation. Cell lines were grown to 80%-90% confluency before harvesting. Both tissues and cell lines were disrupted and extracted in RIPA buffer. An immunoblot of extracts prepared from different rat tissue was performed. A single immunoreactive band at 46 KD was seen in a brain extract. This was in agreement with the predicted size of the polypeptide encoded by PREl cDNA isolated from the mouse brain library. A similar 46 KD band was also seen in other tissues including lung and testis. In addition, however, prominent immunoreactivity to bands at other molecular weights was seen in most tissues, and some tissues lacked a 46 KD band entirely (e.g., skeletal muscle, heart, spleen and liver) . All tissues except brain, showed a major 65 KD band, and two bands around 55 KD were also seen in lung, spleen, testis and liver. The 65 and 55 KD bands may represent isoforms of PREl, the existence of which is suggested by the partial cDNAs isolated from a variety of cDNA libraries. The anti-PREl antibody also immunoblotted a single polypeptide in an extract prepared from C. elegans . This band was approximately 74 KD. The murine brain PREl cDNA was tagged at the PREl amino terminus with an hemagglutinin (HA) epitope and expressed transiently in COS cells. In an immunoblot with anti-PREl antibodies, HA-PRE1 showed the expected size of 46 KD. Extracts prepared from several cell line were subjected to PREl immunoblot. Of the cell lines examined, only BC3H1, a vascular smooth muscle-like line derived from a radiation induced murine brain tumor, showed a single band at 46 KD. A band of similar size was seen in several other cell lines including RIE-1 (rat intestinal epithelial) , MCF-7 (human breast cancer) , HEK 293 (human embryonic kidney) and KB (human oral carcinoma) . Immunoreactive polypeptides of 55 KD (RIE-1, MCF-7, HEK 293 and KB) and 65 KD (RIE-1, HEK 293 and KB), were at least as abundant in these cell lines, and some lines showed only bands other than the 46 KD polypeptide (e.g., Huh-7, 40 KD; L6, 55 KD) . Ras/PRE binding in vi tro A GST-PRE1 (a. a. 188-413) fusion protein (corresponding to the PREl polypeptide encoded by the initial cDNA isolate) was expressed and purified from E. coli . Purified, prokaryotic recombinant [c-H-Ras] (2.5 mg/ml) was loaded with [GTP-γ-S] (2 mM) or [GDP- / 3-S] (2 mM) at 37°C for 15 minutes in the buffer containing 50 mM Tris-HCl, pH 7.5, 7.5 mM EDTA, 2.5 mM MgCl 2 , 0.5 mg/ml bovine serum albumin and 1 mM DTT. Various amounts of GTP-γ-S or GDP- / 3-S loaded Ras proteins were mixed with purified prokaryotic recombinant GST-PRE (188-413) or GST as a control. After incubation at 30°C for 20 minutes, 0.4 ml of 7.5% (V/V) glutathione-Sepharose™ beads suspended in the binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl , 1% Triton X-100, 0.2% BSA and 2 mM DTT), was added to recover GST or GST fusion proteins and any associated proteins. After tumbling at 4°C for 30 minutes, the beads were washed five times with the binding buffer and the bound polypeptides were eluted with SDS sample buffer. The proteins were separated by SDS-PAGE, transferred to PVDF membrane and probed with the monoclonal anti-Ras Pan-Ras-2 antibodies (Oncogene Science) . The bands were visualized using the ECL reagents (Amersham) . This assay showed that GST-PRE1 (a. a. 188-413) , but not GST, binds to Ras, and considerably more Ras-GTP-γ-S bound than Ras-GDP-β-S . These results showed the direct binding of PREl and Ras proteins, and established the binding of the two proteins was GTP-dependent. Ras-PREl association in COS-7 cells The following experiment was carried out using the COS-7 transient expression system. This system was used because it was previously shown that in serum-starved COS-7 cells, Ras is mostly in the GDP bound form, and addition of epidermal growth factor (EGF) or tetradecanoyl phorbol acetate (TPA) rapidly increases Ras-GTP charging (McCollam et al . , 1997, J " . Biol . Chem. , 270:15954-15947) . COS-7 cells were plated at a density of 1.2 million per 10 cm dish and transfected 24 hours later with 7 μg of pMT2-HA-c-H-Ras (Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manual , (2nd Edition) , Cold Spring Harbor Press, Cold Spring Harbor, NY) or empty vector and 12 μg of pEBG-GST-PREl (Nagata et al . , 1990, Nucleic Acids Research, 18:5322) using the DEAE- Dextran method. Forty-eight hours later, cells were starved for 24 hours and subsequently were stimulated with of EGF (100 ng/ml) or TPA (0.1 μM) for various times. Cells were extracted in lysis buffer (30 mM HEPES, pH 7.4, 1% Triton X-100, 20 mM /3-glycerophosphate, 2 mM NaPPi, 1 mM orthovanadate, 20 mM NaF, 20 mM KCl , 2 mM EGTA, 3 mM EDTA, 7.5 mM MgCl 2 , 14 mM /3-ME and a cocktail of protease inhibitors) . Lysates were freeze-thawed once and spun at 17000 X g for 20 minutes. Supernatants were incubated with monoclonal anti-HA antibodies (12CA5) and protein A-G Sepharose™ beads for 3-4 hours at 4°C and then washed extensively with lysis buffer. The washed beads were eluted in SDS sample buffer. The extracted proteins were subjected to SDS-PAGE, transferred on PVDF membranes and probed using affinity-purified anti-GST polyclonal antibodies or monoclonal anti-HA antibodies. Bound antibodies were visualized using ECL. This assay showed that GST-PRE1 was specifically bound by HA-c-H-Ras, but only after the cells were treated with EGF or TPA. The expression of HA-c-H-Ras and of GST-PRE1 was uniform throughout. Thus, PREl was not detectably associated with Ras in serum-starved COS cells, however, within 5 minutes after stimulation by EGF (or TPA) , PREl associated specifically with Ras. This association diminished by 15 minutes after EGF addition, and was largely reversed by 40 minutes. This may have reflected the downregulation of Ras activation after EGF treatment. Ras-PRE association in KB cells In si tu association between endogenous Ras and endogenous PREl, under conditions where the levels of the two polypeptides were not increased artificially by transient overexpression, was examined. The human oral carcinoma cell line, KB, expressed both a readily detectable level of PREl as well as a substantial number of EGF receptors. KB cells were grown to 80% confluency, starved of serum for 24 hours and subsequently stimulated with EGF (100 ng/ml) for various times. Triton X-100 soluble cell lysates were subjected to immunoprecipitation using the monoclonal anti -Ras antibody, Y13-238, which is known to enable isolation of Ras-effector complexes. The Ras immunoprecipitates were washed extensively with the lysis buffer, eluted in SDS sample buffer and subjected to SDS-PAGE, transferred to PVDF membrane and immunoblotted with the affinity purified polyclonal anti-PREl antibodies. These experiments showed that, although equal amounts of endogenous Ras was recovered in all samples, the Ras immunoprecipitates contain immunoreactive PREl only after treatment of the cells with EGF. The time course of Ras-PREl association after EGF treatment in KB cells was more sustained than that observed in COS-7 cells. This may have reflected a different time course of downregulation of Ras activation in those cells. The 46 KD immunoreactive PREl polypeptide was recovered with c- Ras, but not the equally abundant 55 KD protein. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: The General Hospital Corporation (ii) TITLE OF THE INVENTION: RAS-BINDING PROTEIN (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fish & Richardson, P.C. (B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: US (F) ZIP: 02110-2804 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: Windows95 (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 01-OCT-1998 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/942 , 572 (B) FILING DATE: Ol-OCT-1997 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Fraser, Janis K. (B) REGISTRATION NUMBER: 34,819 (C) REFERENCE/DOCKET NUMBER: 08472/737001 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 617/542-5070 (B) TELEFAX: 617/542-8906 (C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3018 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION: 31...1269 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GTAGCTGCGC CGCTGACTGA GGCCTTGGCC ATG GCT TCC CCG GCC ATC GGG CAA 54 Met Ala Ser Pro Ala He Gly Gin 1 5 CGT CCC TAC CCG CTG CTC CTA GAT CCC GAG CCG CCG CGG TAT CTG CAG 102 Arg Pro Tyr Pro Leu Leu Leu Asp Pro Glu Pro Pro Arg Tyr Leu Gin 10 15 20 AGT CTG GGT GGC ACC GAG CCG CCA CCT CCC GCC CGG CCG CGC CGC TGC 150 Ser Leu Gly Gly Thr Glu Pro Pro Pro Pro Ala Arg Pro Arg Arg Cys 25 30 35 40 ATC CCC ACG GCC CTG ATC CCC GCG GCC GGG GCG TCA GAG GAT CGC GGT 198 He Pro Thr Ala Leu He Pro Ala Ala Gly Ala Ser Glu Asp Arg Gly 45 50 55 GGC AGG AGG AGT GGC CGG AGG GAC CCC GAA CCC ACG CCC CGA GAC TGC 246 Gly Arg Arg Ser Gly Arg Arg Asp Pro Glu Pro Thr Pro Arg Asp Cys 60 65 70 CGA CAC GCT CGC CCT GTC CGG CCC GGT CTG CAG CCG AGA CTG CGG CTG 294 Arg His Ala Arg Pro Val Arg Pro Gly Leu Gin Pro Arg Leu Arg Leu 75 80 85 CGA CCT GGG TCA CAC CGA CCC CGC GAC GTG AGG AGC ATC TTC GAG CAG 342 Arg Pro Gly Ser His Arg Pro Arg Asp Val Arg Ser He Phe Glu Gin 90 95 100 CCG CAG GAT CCC CGC GTC TTG GCC GAG AGA GGC GAG GGG CAC CGT TTC 390 Pro Gin Asp Pro Arg Val Leu Ala Glu Arg Gly Glu Gly His Arg Phe 105 110 115 120 GTG GAA CTG GCG CTG CGG GGC GGT CCG GGC TGG TGT GAC CTG TGC GGA 438 Val Glu Leu Ala Leu Arg Gly Gly Pro Gly Trp Cys Asp Leu Cys Gly 125 130 135 CGA GAG GTG CTG CGG CAG GCG CTG CGC TGC GCT AAT TGT AAA TTC ACC 486 Arg Glu Val Leu Arg Gin Ala Leu Arg Cys Ala Asn Cys Lys Phe Thr 140 145 150 TGC CAC TCG GAG TGC CGC AGC CTG ATC CAG TTG GAC TGC AGA CAG AAG 534 Cys His Ser Glu Cys Arg Ser Leu He Gin Leu Asp Cys Arg Gin Lys 155 160 165 GGG GGC CCT GCC CTG GAT AGA CGC TCT CCA GGA AGC ACC CTC ACC CCA 582 Gly Gly Pro Ala Leu Asp Arg Arg Ser Pro Gly Ser Thr Leu Thr Pro 170 175 180 ACC TTG AAC CAG AAT GTC TGT AAG GCA GTG GAG GAG ACA CAG CAC CCG 630 Thr Leu Asn Gin Asn Val Cys Lys Ala Val Glu Glu Thr Gin His Pro 185 190 195 200 CCC ACG ATA CAG GAG ATC AAG CAG AAG ATT GAC AGC TAT AAC AGC AGG 678 Pro Thr He Gin Glu He Lys Gin Lys He Asp Ser Tyr Asn Ser Arg 205 210 215 GAG AAG CAC TGC CTG GGC ATG AAG CTG AGT GAA GAT GGC ACC TAC ACA 726 Glu Lys His Cys Leu Gly Met Lys Leu Ser Glu Asp Gly Thr Tyr Thr 220 225 230 GGT TTC ATC AAA GTG CAT TTG AAG CTC CGA CGG CCA GTG ACG GTG CCC 774 Gly Phe He Lys Val His Leu Lys Leu Arg Arg Pro Val Thr Val Pro 235 240 245 GCT GGA TCC GGC CCC AGT CCA TCT ATG GAT GCC ATT AAG GAA GTG AAC 822 Ala Gly Ser Gly Pro Ser Pro Ser Met Asp Ala He Lys Glu Val Asn 250 255 260 CCT GCA GCC ACC ACA GAC AAG CGG ACT TCC TTC TAC CTG CCA CTC GAT 870 Pro Ala Ala Thr Thr Asp Lys Arg Thr Ser Phe Tyr Leu Pro Leu Asp 265 270 275 280 GCC ATC AAG CAG CTA CAT ATC AGC AGC ACC ACC ACG GTT AGT GAG GTC 918 Ala He Lys Gin Leu His He Ser Ser Thr Thr Thr Val Ser Glu Val 285 290 295 ATC CAG GGG CTG CTC AAG AAG TTC ATG GTT GTG GAC AAC CCA CAG AAG 966 He Gin Gly Leu Leu Lys Lys Phe Met Val Val Asp Asn Pro Gin Lys 300 305 310 TTT GCA CTT TTT AAG CGG ATA CAC AAA GAT GGA CAA GTG CTC TTC CAG 1014 Phe Ala Leu Phe Lys Arg He His Lys Asp Gly Gin Val Leu Phe Gin 315 320 325 AAA CTC TCC ATT GCT GAC TAT CCT CTC TAC CTT CGT CTG CTC GCT GGG 1062 Lys Leu Ser He Ala Asp Tyr Pro Leu Tyr Leu Arg Leu Leu Ala Gly 330 335 340 CCT GAC ACC GAT GTT CTC AGC TTT GTG CTA AAG GAG AAT GAA ACT GGA 1110 Pro Asp Thr Asp Val Leu Ser Phe Val Leu Lys Glu Asn Glu Thr Gly 345 350 355 360 GAG GTG GAG TGG GAT GCC TTT TCC ATT CCT GAA CTC CAG AAC TTT TTA 1158 Glu Val Glu Trp Asp Ala Phe Ser He Pro Glu Leu Gin Asn Phe Leu 365 370 375 ACT ATC CTG GAA AAA GAG GAG CAG GAC AAG ATC CAT CAA CTG CAA AAG 1206 Thr He Leu Glu Lys Glu Glu Gin Asp Lys He His Gin Leu Gin Lys 380 385 390 AAG TAC AAC AAA TTC CGT CAG AAA CTG GAA GAG GCA TTA CGA GAG TCC 1254 Lys Tyr Asn Lys Phe Arg Gin Lys Leu Glu Glu Ala Leu Arg Glu Ser 395 400 405 CAA GGG AAG CCG GGG TAACCAGCCG ACTTCCTGTC CTCTCAGTGC CCTCCAATTT 1309 Gin Gly Lys Pro Gly 410 ATTTTATTGT TAATTATTTT GCAACAAAGA GTTACTGTTA AGACACCTCT GGTGGTTCCA 1369 CCAGTCGCCT GCCCAGCAGT TAACAGATGT GGCACAAAGT CTCTTCCACG CAGTGTCTAT 1429 GCAGGGTTCC GATTCCTGCT AACCCACCAC ACCATGGCTC TGGAGAGCTT CCCGCCTGGG 1489 ATCAGAACTC CTGTGGAATG ACCAGTGTTT CCCTGCTCAG TCTGCTGGCC TCTCAGAAAC 1549 CAAATAGTTG CCTCTCTGGT CACCAAACTC CAATCAATCA CCAGCCGGCA AAAGGAAAGA 1609 AAGGTTTCAG AGCCTGTGTG TTCTTTCTCT GGATTTACTC TTCAGTTCCT CTTTTGGTTT 1669 GTTTGGTTGG TTTTTTTTGG CCACGTATAG TATATTTAAG GATCAAATGT GGCATATTCA 1729 TTCTAGCTAA GTCCTTGAAA GCAGGAAAAT GCTCATGAAA GGACTGTCCT TGCCCCAAGG 1789 TGCCTCTTCT TCTCTAGTAC TAGACACTCA GGGTCAGCCT GAGATTTCAA GAGGCTACAG 1849 CCTGACCAGG CCGTCTTCTT ATTACCCAGC AGGCTGTGTG CATGCAAACC CAAAGACATA 1909 TATGCACATC TGTGTGGTAT TTCAGCATGT CTCTGTCCAA TGTTTGATAT GTTAACATTT 1969 GAATTTAATG CTGTCCTCCT TATGGGTTTC TACCAAAGAG AAACCAGCCA CTTATCAATT 2029 TTAGTTTCTT GCTGAGCTGC CAGAAAGTAT TACAGAGAAG CACATCCAAG CTGTCTGTGG 2089 CCTACGCCTG CAGGGGGTGG GGGGCCTGAA TCTCCTTGGC CTTCAGTTCC ACCTCCACCT 2149 CTGGCTTTAG GGTCTCCAGC TGTTGCCTGA GTAGTAGCTT TGATTACAGC GGTAAAGTCC 2209 TCCAACTTGG AGTCCTTTCT GGTGGGAAGC ATGGTCTGCT CGCAGCACAG CACTGAGCAG 2269 ACCCGTGGGC CTGACTTCCC TGGTGACTTC AGTGCCTTTT TGTTTGCAGA GAAAAGAGTG 2329 GGGCACTTTG CTTGAAGCTC TCTGCTGGCT TGCCCCTGGC AGGAAGTGGA CAATGGTGCT 2389 ATAGAGCCAA GGACACAGCC TCAGAGCACA GGGTGATTGA TGATCAGCCT CTTTCCCATC 2449 AAGCTTCCCG GTCAGGCTTT GACTTTGAAG ATGCGAGGTT ACTAGACTGC ATTGACAGCA 2509 TCAGATTATG ACTCCAACTC TTGAGTAGTT CAGACTTAAA ACCAATCAGC CAGAGTAGCC 2569 AGGACTGCAA AGACACTCAA TACAGATGGA GAAAAACTTG TCCCTTTAAA AGAGGGCCAG 2629 TGTTTCAATT GAGCCTCCAG AGGAGACCAC TTTCATGTTG TGCTTGCCTT TCCATACCCT 2689 TTCCTCGGGT TGTTTTAAGC CCAAGCTTCT CCGTGTAGCC TAAAAAGTTC CCTACCAGCC 2749 CAGCTGAAGC CACACTGCTC CCGTCCCAGA AGAACGCCAA ATCCTTGTCA TTCAAACTGT 2809 GCATCGTTTG CAGAGCTGCA AAAAGCAACA TGAGCTAGCG ACTCTGAGGT TGTGCACGCC 2869 ATCAGCCCCT TGGCTGCCTG AGGTCTCATG CCCAGCCTTA CACCTCTCTC CCTTAAGAAG 2929 CCCCCGTCCT GCTGTGTACT ACAGGGGCAC GTGGAATCAT TCCCTTCATC CTGCATGTCT 2989 GTAGCGTTAG GAGAAGGCAT GGCTCCTGC 3018 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 413 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Ser Pro Ala He Gly Gin Arg Pro Tyr Pro Leu Leu Leu Asp 1 5 10 15 Pro Glu Pro Pro Arg Tyr Leu Gin Ser Leu Gly Gly Thr Glu Pro Pro 20 25 30 Pro Pro Ala Arg Pro Arg Arg Cys He Pro Thr Ala Leu He Pro Ala 35 40 45 Ala Gly Ala Ser Glu Asp Arg Gly Gly Arg Arg Ser Gly Arg Arg Asp 50 55 60 Pro Glu Pro Thr Pro Arg Asp Cys Arg His Ala Arg Pro Val Arg Pro 65 70 75 80 Gly Leu Gin Pro Arg Leu Arg Leu Arg Pro Gly Ser His Arg Pro Arg 85 90 95 Asp Val Arg Ser He Phe Glu Gin Pro Gin Asp Pro Arg Val Leu Ala 100 105 110 Glu Arg Gly Glu Gly His Arg Phe Val Glu Leu Ala Leu Arg Gly Gly 115 120 125 Pro Gly Trp Cys Asp Leu Cys Gly Arg Glu Val Leu Arg Gin Ala Leu 130 135 140 Arg Cys Ala Asn Cys Lys Phe Thr Cys His Ser Glu Cys Arg Ser Leu 145 150 155 160 He Gin Leu Asp Cys Arg Gin Lys Gly Gly Pro Ala Leu Asp Arg Arg 165 170 175 Ser Pro Gly Ser Thr Leu Thr Pro Thr Leu Asn Gin Asn Val Cys Lys 180 185 190 Ala Val Glu Glu Thr Gin His Pro Pro Thr He Gin Glu He Lys Gin 195 200 205 Lys He Asp Ser Tyr Asn Ser Arg Glu Lys His Cys Leu Gly Met Lys 210 215 220 Leu Ser Glu Asp Gly Thr Tyr Thr Gly Phe He Lys Val His Leu Lys 225 230 235 240 Leu Arg Arg Pro Val Thr Val Pro Ala Gly Ser Gly Pro Ser Pro Ser 245 250 255 Met Asp Ala He Lys Glu Val Asn Pro Ala Ala Thr Thr Asp Lys Arg 260 265 270 Thr Ser Phe Tyr Leu Pro Leu Asp Ala He Lys Gin Leu His He Ser 275 280 285 Ser Thr Thr Thr Val Ser Glu Val He Gin Gly Leu Leu Lys Lys Phe 290 295 300 Met Val Val Asp Asn Pro Gin Lys Phe Ala Leu Phe Lys Arg He His 305 310 315 320 Lys Asp Gly Gin Val Leu Phe Gin Lys Leu Ser He Ala Asp Tyr Pro 325 330 335 Leu Tyr Leu Arg Leu Leu Ala Gly Pro Asp Thr Asp Val Leu Ser Phe 340 345 350 Val Leu Lys Glu Asn Glu Thr Gly Glu Val Glu Trp Asp Ala Phe Ser 355 360 365 He Pro Glu Leu Gin Asn Phe Leu Thr He Leu Glu Lys Glu Glu Gin 370 375 380 Asp Lys He His Gin Leu Gin Lys Lys Tyr Asn Lys Phe Arg Gin Lys 385 390 395 400 Leu Glu Glu Ala Leu Arg Glu Ser Gin Gly Lys Pro Gly 405 410

Claims: Claims We claim: 1. An isolated DNA comprising a nucleotide sequence that encodes a naturally-occuring protein that: (a) shares at least 80% sequence identity with SEQ ID NO: 2 and (b) binds to Ras. 2. The DNA of claim 1, wherein said nucleotide sequence defines a DNA molecule whose complement hybridizes under high stringency conditions to a DNA whose nucleotide sequence consists of SEQ ID NO : 1. 3. The DNA of claim 1, wherein said protein has an amino acid sequence consisting of SEQ ID NO: 2. 4. The DNA of claim 3, wherein said nucleotide sequence is the coding region of the nucleotide sequence of SEQ ID N0:1. 5. A vector comprising the DNA of claim 1. 6. The vector of claim 5, wherein said DNA is operably linked to one or more expression control sequences . 7. A cell comprising the DNA of claim 1. 8. A substantially pure protein that: (a) comprises an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO: 2 and (b) binds to Ras. 9. A substantially pure protein comprising amino acid residues 266-360 of SEQ ID NO:2. 10. A substantially pure protein comprising amino acid residues 188-413 of SEQ ID NO:2. 11. A substantially pure protein whose amino acid sequence is SEQ ID NO: 2. 12. The protein of claim 9, wherein the protein is a fused heterologous polypeptide. 13. The protein of claim 12, wherein said fused heterologous polypeptide comprises a glutathione-S- transferase fragment. 14. An antibody that binds specifically to the protein of claim 8. 15. The antibody of claim 10, further comprising a detectable label. 16. A screening method for identifying a substance that modulates binding of a PREl protein to a Ras protein, said method comprising the steps of: (a) providing a sample solution of a PREl protein; (b) adding to said sample solution a candidate substance; (c) adding to said sample solution a Ras protein; and (d) detecting an increase or decrease in binding of said PREl protein to said Ras protein in the presence of said candidate substance, compared to the binding of the PREl protein to the Ras protein in the absence of said candidate substance, as an indication that said candidate substance modulates binding of said PREl protein to said Ras protein. 17. A method of producing a PREl protein, said method comprising the steps of : (a) providing a cell transformed with an isolated DNA comprising a nucleotide sequence that encodes a PREl protein; (b) culturing said cell; and (c) collecting said PREl protein encoded by said nucleotide sequence. 18. A screening method for identifying a substance that modulates PREl gene expression, said method comprising the steps of : (a) providing a test cell; (b) contacting said test cell with a candidate substance; and (c) detecting an increase or decrease in the level of PREl gene expression in the presence of said candidate substance, compared to the level of PREl gene expression in the absence of said candidate substance, as an indication that said candidate substance modulates PREl gene expression. 19. A method for isolating a PREl-binding substance, said method comprising the steps of: (a) providing a sample of an immobilized PREl protein; (b) contacting a mixture containing said PREl- binding substance with said immobilized PREl protein; (c) separating unbound components of said mixture from bound components of said mixture; (d) recovering said PREl-binding substance from said immobilized PREl protein. 20. The method of claim 15, wherein said PREl- binding substance is Ras protein.

Inventors: AVRUCH JOSEPH, ZHANG XIAN-FENG, AVRUCH, JOSEPH, ZHANG, XIAN-FENG
Applicants: AVRUCH JOSEPH, GEN HOSPITAL CORP, ZHANG XIAN FENG, AVRUCH, JOSEPH, THE GENERAL HOSPITAL CORPORATION, ZHANG, XIAN-FENG
Application year: 1998
Main classification: C07H21
IPC codes: A61K38, A61K45, A61P35, A61P43, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33