JP2001516586A - DNA encoding RAS carboxyl-terminal processing protein - Google Patents

Abstract

  (57) [Summary] DNA encoding the human RAS carboxyl-terminal processing protein was cloned and characterized. A recombinant protein can form a biologically active protein. The cDNAs are expressed in recombinant host cells, which produce active recombinant proteins. Further, the recombinant protein is purified from the recombinant host cell. In addition, recombinant host cells are utilized to establish methods for identifying modulators of enzymatic activity, and modulators are identified.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

Ras protein acts as a major regulator of cell growth and differentiation 21 kD
a. Guanosine triphosphate (GTP) binding protein. It is an active GT
Circulates between the P-linked form and the inactive GDP form. Ras, when constitutively active due to a mutation that anchors the protein to its GTP state, causes tumorigenesis (
1). Ras is found in 50% of colon cancers and 95% of pancreatic cancers (
2). Ras becomes the membrane-bound necessary to exert its biological effects upon three post-translational carboxyl-terminal modifications (3). The Ras protein ends with a CAAX motif, where C is cysteine, A is an aliphatic amino acid, and X is any amino acid. The first step is the addition of the lipid component farnesyl at the cysteine residue via a thioether bond. This is followed by removal of the three COOH-terminal amino acids and the addition of a methyl group to the newly exposed carboxylprenylcysteine. These three modifications are essential for transformation activity. A yeast gene has been identified that results in the removal of three terminal amino acids (4). Deletion of the gene has been shown to interfere with ras function, ie, ras localization and signal transduction. Described herein is the identification of a novel human ras protease isolated by homology search against the yeast amino acid sequence. Human ra
Inhibition of s protease should be effective for cancer treatment.

[0002] Using nested PCR technology, a cDNA sequence believed to correspond to the human ras protease gene was cloned from a human colon adenocarcinoma cDNA library. The sequence is a protease that leads to ras processing in yeast (4)
Has 41.1% nucleotide identity to yeast RCE1. It is 1348b
p, about 1.35 k in Northern blot of RNA from various cancer tissues.
hybridizes to the mRNA transcript of b. In a search of the nucleotide and amino acid sequences of our clones and GeneBank, the yeast RCE1 was elicited as the first hit. All these results support the idea that we have indeed cloned the human ras protease.

[0003]

[Table 1]

[0004]

SUMMARY OF THE INVENTION

The DNA molecule encoding the ras carboxyl-terminal processing protein has been cloned and characterized, and represents a novel class of ras processing enzymes. Using a recombinant expression system, a functional DNA molecule encoding the ras carboxyl-terminal processing protein was isolated. Disclose the biological and structural properties of these proteins, as well as amino acid and nucleotide sequences. The recombinant protein is r
Useful for identifying modulators of as carboxyl-terminal processing proteins. Modulators identified in the assays disclosed herein are useful as therapeutics. The recombinant DNA molecule and portions thereof may be used to isolate homologs of the DNA molecule, identify and isolate genomic equivalents of the DNA molecule, and identify and detect mutant forms of the DNA molecule, or Useful for isolation.

[0005]

[Detailed description]

The present invention relates to a DNA encoding a ras carboxyl-terminal processing protein isolated from cells producing ras carboxyl-terminal processing protein. ra
An s-carboxyl-terminal processing protein, as used herein,
Refers to a protein that can specifically function as a ras modulator.

[0006] The complete amino acid sequence of the human ras carboxyl-terminal processing protein has previously been described.
And a human ras carboxyl-terminal processing protein
The complete nucleotide sequence to be sequenced was not known. This is ras carboxy
First reported clone of a full-length DNA molecule that encodes
Learning. Ras carboxyl terminus described by a wide variety of cells and cell types
Predicted to contain processing protein. ras carboxyl terminal process
Vertebrate cells capable of producing signaling proteins include poly-A isolated from the cells. ⁺ As shown by the ability of the ras carboxyl-terminal processing protein cDNA to hybridize to RNA: heart, brain, liver, skeletal muscle, placenta, lung, kidney
And the pancreas. In addition, ras carboxyl-terminal processing protein RNA
HL-60, HeLa, K562, Molt-4, Burkitri as described above.
Emphysema Raji, colon adenocarcinoma, SW480, lung cancer A549 and melanoma, G361
Detected in primary human cancer cell lines.

[0007] Other cells and cell lines also use the ras carboxyl-terminal processing protein cD.
It may be suitable for use to isolate NA. Ra in cell extracts
Appropriate cells can be selected by screening for s-carboxyl-terminal processing protein activity or by Northern analysis to detect mRNA.
Activity can be assessed by detecting the processing activity on the ras carboxyl terminal substrate. Cells that possess ras carboxyl-terminal processing protein activity in this assay may be suitable for the isolation of ras carboxyl-terminal processing protein DNA or mRNA.

[0008] Any of a variety of methods known in the art may be used to molecularly clone ras carboxyl-terminal processing protein DNA.
These methods include, but are not limited to, constructing a cDNA library containing the ras carboxyl-terminal processing protein in a suitable expression vector system followed by direct functional expression of the ras carboxyl-terminal processing protein gene. Another method is to screen a cDNA library containing a ras carboxyl-terminal processing protein constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe designed from the amino acid sequence of the ras carboxyl-terminal processing protein subunit. is there. A further method comprises screening a cDNA library containing the ras carboxyl-terminal processing protein constructed in a bacteriophage or plasmid shuttle vector with the partial cDNA encoding the ras carboxyl-terminal processing protein. This partial cDNA is obtained by specific PCR amplification of the ras carboxyl-terminal processing protein DNA fragment by designing degenerate oligonucleotide primers from the amino acid sequence of the purified ras carboxyl-terminal processing protein.

Another method uses RNA from ras carboxyl-terminal processing protein-producing cells.
And translating the RNA into a protein by an in vitro or in vivo translation system. Translation of the RNA into a peptide or protein results in the production of at least a portion of the ras carboxyl-terminal processing protein, which can
It can be identified by immunological reactivity with the ras carboxyl-terminal processing protein antibody or by the biological activity of the ras carboxyl-terminal processing protein. In this way, a pool of RNA isolated from ras carboxyl-terminal processing protein producing cells can be analyzed for the presence of RNA encoding at least a portion of the ras carboxyl-terminal processing protein. r
Further fractionation of the RNA pool can be performed to purify ras carboxyl-terminal processing protein RNA from RNA other than as carboxyl-terminal processing protein. The amino acid sequence can be determined by analyzing the peptides or proteins produced by this method, and then used to provide primers to generate the ras carboxyl-terminal processing protein cDNA, or to analyze the RNA used for translation. Thus, a nucleotide sequence encoding the ras carboxyl-terminal processing protein can be provided, and a probe for generating the ras carboxyl-terminal processing protein cDNA can be prepared. This method is known in the art and is described, for example, in Maniatis, T .; , Frit
sch, E.C. F. Sambrook, J .; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

It is readily apparent to one of skill that other types of libraries and libraries constructed from other cells or cell types may be useful for isolating DNA encoding the ras carboxyl-terminal processing protein. Is obvious. Other types of libraries include cDNA libraries obtained from other cells, other organisms than humans, and genomic DNA, including YAC (yeast artificial chromosome) and cosmid libraries.
Including, but not limited to, NA libraries.

It is readily apparent to those skilled in the art that a suitable cDNA library can be prepared from cells or cell lines that have ras carboxyl-terminal processing protein activity. The selection of cells or cell lines used to prepare a cDNA library for isolating the ras carboxyl-terminal processing protein cDNA by first measuring the ras carboxyl-terminal processing protein activity associated with the cells using the ras carboxyl-terminal substrate. Can be implemented.

[0012] Preparation of a cDNA library can be performed by standard techniques well known in the art. See, for example, Maniatis, T .; , Fritsch,
E. FIG. F. Sambrook, J .; , Molecular Cloning: A
Well-known cDNA library construction techniques can be found in the Laboratory Manual, 2nd edition, (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

It is also readily apparent to one skilled in the art that the DNA encoding the ras carboxyl terminal processing protein may be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. See, for example, Maniatis, T .; , Fritsch
, E .; F. Sambrook, J .; , Molecular Cloning: A Laboratory Manual, 2nd edition, (Cold Spring H
A well-known genomic DNA library construction technique can be found in the Arbor Laboratory, Cold Spring Harbor, NY, 1989).

[0014] In order to clone the ras carboxyl terminal processing protein gene by the above method, the amino acid sequence of the ras carboxyl terminal processing protein may be required. To accomplish this, the ras carboxyl-terminal processing protein can be purified and the partial amino acid sequence determined using an automatic sequencer. Although it is not necessary to determine the entire amino acid sequence, a linear sequence of two regions of six to eight amino acids from the protein is determined to prepare primers for PCR amplification of the partial ras carboxyl-terminal processing protein DNA fragment.

[0015] Once suitable amino acid sequences have been identified, DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, one or more codons can be used to encode a particular amino acid, and therefore a set of similar D
The amino acid sequence can be encoded by any of the NA oligonucleotides. Only one member of the set is identical to the ras carboxyl-terminal processing protein sequence, but even in the presence of mismatched DNA oligonucleotides can hybridize to the ras carboxyl-terminal processing protein DNA. The DNA oligonucleotide having the mismatch may still hybridize sufficiently to the ras carboxyl-terminal processing protein DNA, identifying the DNA encoding the ras carboxyl-terminal processing protein,
Can be isolated. DN from various cell types, from invertebrate and vertebrate sources
DNA isolated by these methods can be used to screen the A library and to isolate homologous genes.

[0016] The purified biologically active ras carboxyl-terminal processing protein can take several different physical forms. The ras carboxyl-terminal processing protein can be present as a full-length nascent or unprocessed polypeptide, or as a partially processed polypeptide or a combination of processed polypeptides. The full-length nascent ras carboxyl-terminal processing protein polypeptide can be post-translationally modified by a specific proteolytic cleavage event, which results in the formation of fragments of the full-length nascent nascent polypeptide. Fragments or the physical association of fragments may have the full biological activity associated with the ras carboxyl-terminal processing protein; however, the extent of ras carboxyl-terminal processing protein activity is dependent on the individual ras carboxyl-terminal processing protein fragment and the physical It is possible that ras carboxyl-terminal processing protein polypeptide fragments that are specifically associated may differ.

[0017] Recombinant by cloning the cloned ras carboxyl-terminal processing protein DNA obtained by the method described herein into an expression vector containing a suitable promoter and other suitable transcriptional regulatory elements. And can be introduced into prokaryotic or eukaryotic host cells to produce recombinant ras carboxyl-terminal processing protein.
Techniques for such manipulation are described in Maniatis, T .; Etc., are fully described above and are well known in the art.

[0018] An expression vector is defined herein as a DNA sequence necessary for the transcription of cloned copies of the gene and translation of their mRNA in a suitable host. Escherichia coli (E. Coli) bacteria including the, cyanobacteria, plant cells, insect cells, in order to express eukaryotic genes in a variety of hosts such as fungal cells and animal cells including yeast cells Such vectors can be used.

Particularly designed vectors allow DNA to be shuttled between hosts such as bacterial-yeast or bacterial-animal cells or bacterial-fungal cells or bacterial-invertebrate cells. A properly constructed expression vector should contain: an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, a high copy number potential and an active promoter. . A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates mRNA at a high frequency. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, particularly designed plasmids or viruses.

A variety of mammalian expression vectors can be used to express the recombinant ras carboxyl-terminal processing protein in mammalian cells. Recombination ra
Commercially available mammalian expression vectors that may be suitable for s-carboxyl-terminal processing protein expression include pMAMneo (Clontech), pcD
NA3 (Invitrogen), pMC1neo (Stratagene),
pXT1 (Stratagene), pSG5 (Stratagene), EB
O-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC
37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCT
ag (ATCC 37460) and IZD35 (ATCC 37565).

[0021] A variety of bacterial expression vectors can be used to express the recombinant ras carboxyl-terminal processing protein in bacterial cells. Commercially available bacterial expression vectors that may be suitable for recombinant ras carboxyl-terminal processing protein expression include pET vectors (Novagen).
), PQE vector (Qiagen), pGEX (Pharmacia) and pQE
TrcHis (Invitrogen), but is not limited thereto.

A variety of fungal cell expression vectors can be used to express the recombinant ras carboxyl-terminal processing protein in a fungal cell, such as yeast.
A commercially available fungal cell expression vector that may be suitable for recombinant ras carboxyl-terminal processing protein expression is pYES2 (Invitrog
en) and Pichia expression vectors (Invitrogen).

Various insect cell expression vectors can be used to express the recombinant ras carboxyl terminal processing protein in insect cells. A commercially available insect cell expression vector that may be suitable for recombinant expression of the ras carboxyl-terminal processing protein is pBlueBacII (Invitroge).
n), pVL1392 (Pharmingen) and pFastBacHT (G
ibcoBRL).

[0024] DNA encoding the ras carboxyl-terminal processing protein can be cloned into an expression vector for expression in recombinant host cells. Recombinant host cells include bacteria such as Escherichia coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and Drosophila and It may be prokaryotic or eukaryotic, including but not limited to insect cells, including but not limited to silkworm-derived cell lines. Potentially suitable and commercially available cell lines from mammalian species include CV-1 (ATCC CCL 70), COS-1 (COS-1).
ATCC CRL 1650), COS-7 (ATCC CRL 1651), CH
O-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH
/ 3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C
127I (ATCC CRL 1616), BSC-1 (ATCC CCL 26)
, MRC-5 (ATCC CCL 171), L cells and HEK-293 (ATC
C CRL 1573).

The expression vector can be introduced into a host cell by any of a number of techniques including, but not limited to, transformation, transfection, protoplast fusion, lipofection, and electroporation. Cells containing the expression vector are clonally expanded and individually analyzed to determine if they produce a ras carboxyl-terminal processing protein. Immunological reactivity with anti-ras carboxyl-terminal processing protein antibody and ra associated with host cells
The identification of a host cell clone that expresses a ras carboxyl-terminal processing protein can be performed by several methods, including but not limited to the presence of s-carboxyl-terminal processing protein activity.

Alternatively, expression of ras carboxyl-terminal processing protein DNA may be performed using in vitro produced synthetic mRNA. Synthetic mRNA or mRNA isolated from ras carboxyl-terminal processing protein-producing cells can be efficiently translated in various cell-free systems including but not limited to wheat germ extracts and reticulocyte extracts; and Translation can also be performed efficiently in cell-based systems, including but not limited to microinjection into frog oocytes, and microinjection into frog oocytes is generally preferred.

The highest level of ras carboxyl-terminal processing protein activity and / or r
To determine the ras carboxyl-terminal processing protein DNA sequence resulting in an as-carboxyl-terminal processing protein, the following: from about base 6 to about base 1013 (these numbers are the first nucleotide of the first methionine and the first stop codon of the first stop codon) Several constructs containing the full length open reading frame of the ras carboxyl-terminal processing protein cDNA encoding the putative 338 amino acid protein (corresponding to the last nucleotide before) and a portion of the cDNA encoding the ras carboxyl-terminal processing protein Ras carboxyl-terminal processing protein DNA molecules, including but not limited to All constructs can be designed to contain no, all, or a portion of the 5 'or 3' untranslated region of the ras carboxyl-terminal processing protein cDNA. After introducing these constructs, both singly and in combination, into appropriate host cells, the level of ras carboxyl-terminal processing protein activity and protein expression can be determined. After determining the ras carboxyl-terminal processing protein DNA cassette that results in the highest expression in the transient assay, mammalian cells, insect cells infected with baculovirus, Escherichia coli and the yeast S. cerevisiae ( S. cerevisiae )
For expression in host cells, including but not limited to
Transfer the s-carboxyl-terminal processing protein DNA construct to various expression vectors.

[0028] Host cell transfectants and microinjected oocytes can be used to assay both ras carboxyl-terminal processing protein activity levels and ras carboxyl-terminal processing protein levels by the following methods. In the case of recombinant host cells, this involves the co-transfection of one or possibly two or more plasmids containing the ras carboxyl-terminal processing protein DNA encoding one or more fragments or subunits. For oocytes, this is ras
With co-injection of synthetic RNA of carboxyl-terminal processing protein. After a suitable period for expression, the cellular proteins are metabolically metabolized with, for example, ³⁵ S-methionine.
After time labeling, cell lysates and cell culture supernatants are collected and subjected to immunoprecipitation with a polyclonal antibody against the ras carboxyl-terminal processing protein.

Other methods for detecting ras carboxyl-terminal processing protein activity include direct ras processing activity in whole cells transfected with ras carboxyl-terminal processing protein cDNA or oocytes injected with ras carboxyl-terminal processing protein mRNA. Including measurements. The activity of the ras carboxyl-terminal processing protein is determined by specific ras processing of the synthetic ras carboxyl-terminal substrate in extracts of host cells expressing the ras carboxyl-terminal processing protein DNA.

[0030] The level of ras carboxyl-terminal processing protein in host cells is quantified by immunoaffinity and / or ligand affinity techniques. ³⁵ S
-Assaying cells expressing the ras carboxyl-terminal processing protein for the number of expressed ras carboxyl-terminal processing protein molecules by measuring the expression level with a specific antibody against the methionine-labeled or unlabeled ras carboxyl-terminal processing protein. Can be. The labeled ras carboxyl-terminal processing protein is analyzed by SDS-PAGE. ras
Unlabeled ras carboxyl-terminal processing protein is detected by western blotting, ELISA or RIA assay using a carboxyl-terminal processing protein-specific antibody.

[0031] Because the genetic code is degenerate, one or more codons can be used to encode a particular amino acid, thus reducing the amino acid sequence by any of a set of similar DNA oligonucleotides. Can be code. Only one member of the set is identical to the ras carboxyl-terminal processing protein sequence,
Even under the appropriate conditions, the presence of mismatched DNA oligonucleotides
as carboxyl-terminal processing protein DNA. Under other conditions, the DNA oligonucleotide with the mismatch is still ra
has the potential to hybridize to the s-carboxyl-terminal processing protein DNA, identifies the DNA encoding the ras-carboxyl-terminal processing protein,
Can be isolated.

D encoding a ras carboxyl terminal processing protein from a particular organism
NA can be used to isolate and purify ras carboxyl-terminal processing protein homologs from other organisms. To accomplish this, the first r
The as carboxyl-terminal processing protein DNA is converted to Ds encoding a homolog of the ras carboxyl-terminal processing protein under appropriate hybridization conditions.
It can be mixed with a sample containing NA. The hybridized DNA complex can be isolated, and the DNA encoding the homologous DNA can be purified therefrom.

It is known that there is a significant amount of duplication at the various codons that code for a particular amino acid. Therefore, the invention also relates to DNA sequences containing another codon that encodes the final translation of the same amino acid. For the purposes of this specification, sequences that carry one or more substituted codons are defined as degenerate variations. Also included within the scope of the invention are mutations in either the DNA sequence or the translated protein that do not substantially alter the basic physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in polypeptide function.

It is known that the DNA sequence encoding a peptide can be modified to encode a peptide having properties different from those of the naturally occurring peptide. DNA
Methods for altering the sequence include, but are not limited to, site-directed mutagenesis. Examples of modifying properties include, but are not limited to, altering the affinity of the receptor for the enzyme or ligand for the substrate.

As used herein, the ras carboxyl-terminal processing protein “
A “functional derivative” is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of the ras carboxyl-terminal processing protein. The term “functional derivative” is considered to include “fragments”, “variants”, “degenerate variants”, “analogs” and “homologs” or “chemical derivatives” of the ras carboxyl-terminal processing protein. Can be The term "fragment" is considered to refer to any polypeptide subunit of the ras carboxyl-terminal processing protein. The term "variant" is considered to refer to a molecule that is substantially similar in structure and function to either the entire ras carboxyl-terminal processing protein molecule or a fragment thereof. A molecule is "substantially similar" to a ras carboxyl-terminal processing protein if both molecules have substantially similar structures or both molecules possess similar biological activities.
Therefore, if two molecules possess substantially similar activities, even if the structure of one of the molecules is not found in the other, or the amino acid sequences of the two are not the same. , They are considered variants. "Similar"
The term refers to a molecule substantially similar in function to either the entire ras carboxyl-terminal processing protein molecule or a fragment thereof.

After expression of the ras carboxyl-terminal processing protein in the recombinant host cell, the ras carboxyl-terminal processing protein can be recovered to give an active form of the protein. Hitz and Georgopapadakou: [Hitz
A. M. And Georgopapadouko N .; H. (1996) FEBS
Several ras carboxyl-terminal processing protein purification methods are available, including those described by Letsters 391: 310-312].
Suitable for use. To purify ras carboxyl-terminal processing proteins from natural sources as described above, various combinations of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography and hydrophobic interaction chromatography Alternatively, depending on the particular application, recombinant ras carboxyl-terminal processing protein can be purified from cell lysates and extracts or from conditioned culture medium.

In addition, the use of an immunoaffinity column made with a full-length nascent ras carboxyl-terminal processing protein, a polypeptide fragment of the ras carboxyl-terminal processing protein or a monoclonal or polyclonal antibody specific for the ras carboxyl-terminal processing protein subunit. Recombinant ras carboxyl-terminal processing protein can be separated from proteins of other cells. Furthermore, the ras carboxyl terminal processing protein can be expressed as a fusion protein and purified by adsorption to the fusion component, for example, the GST fusion system [Smith, D .; B. And Johnson, K .; S. (19
88) Gene 67:31].

A monospecific antibody against the ras carboxyl-terminal processing protein was identified as ras
Monoclonal antibodies purified from mammalian antisera containing antibodies reactive against carboxyl-terminal processing protein, or reactive with ras carboxyl-terminal processing protein using the technique of Kohler and Milstein, Nature 256: 495-497 (1975). Prepared as As used herein, a monospecific antibody is defined as a single antibody species or multiple antibody species having a uniform binding characteristic of the ras carboxyl-terminal processing protein. Homogeneous binding, as used herein, refers to the ability of an antibody species to bind to a particular antigen or epitope, such as those that bind to the ras carboxyl-terminal processing protein, as described above. Appropriate concentration of ra with or without immune adjuvant
By immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like with the s-carboxyl-terminal processing protein, an antibody specific to the ras-carboxyl-terminal processing protein is prepared, and rabbits are preferred.

Pre-immune serum is collected before the first immunization. Each animal receives between about 0.1 mg and about 1000 mg of the ras carboxyl-terminal processing protein associated with an acceptable immune adjuvant. Such acceptable adjuvants include Freund's complete, Freund's incomplete, alum-precipitate
), Including Corynebacterium parvum (Corynebacterium par vum) water-in-oil emulsions and tRNA containing but not limited to. The initial immunization consists of the ras carboxyl-terminal processing protein in multiple sites, preferably in subcutaneous (SC), intraperitoneal (IP) or both, preferably in complete Freund's adjuvant. Blood is taken from each animal at regular intervals, preferably weekly, to determine antibody titers. Animals may or may not receive booster injections after the first immunization. Animals receiving booster injections generally receive the same amount of antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at approximately 3 week intervals until a maximum titer is obtained. Approximately 7 days after each booster immunization or approximately every week after a single immunization, blood is drawn from the animals, the serum is collected, and aliquots are stored at approximately -20 ° C.

Inbred mice, preferably Bal, with the ras carboxyl-terminal processing protein
A monoclonal antibody (mAb) that reacts with the ras carboxyl terminal processing protein by immunizing b / c is prepared. As described above, about 0.1 ml to about 10 mg, preferably about 1 mg, of the ras carboxyl-terminal processing protein in about 0.5 ml of buffer or saline contained in an equal volume of an acceptable adjuvant is administered by the IP or SC route. To immunize mice. Complete Freund's adjuvant is preferred. Mice are initially immunized on day 0 and rested for about 3 to about 30 weeks. Immunized mice receive one or more booster immunizations of about 0.1 to about 10 mg of ras carboxyl-terminal processing protein in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, preferably spleen lymphocytes, are obtained from antibody-positive mice by removing the spleen from the immunized mouse by standard methods known in the art. Hybridoma cells are prepared by mixing the appropriate fusion partner, preferably myeloid cells, and spleen lymphocytes under conditions that allow stable hybridoma formation. Fusion partners include, but are not limited to, mouse myeloid P3 / NS1 / Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being generally preferred. Antibody-producing cells and myeloid cells were treated with polyethylene glycol, about 1000 mol. wt. Fused in. The fused hybridoma cells are selected by growth in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with hypoxanthine, thymidine and aminopterin by methods known in the art. At about days 14, 18, and 21, supernatants are collected from growth-positive wells and screened for antibody production by immunoassays such as solid-phase immunoradiation assay (SPIRA) using the ras carboxyl-terminal processing protein as antigen. Cultures are also tested in the Ouchterlony precipitation assay to determine the mAb isotype. MacPherson, Sof
t Agar Technologies, Tissue Culture Metho
Hybridoma cells from antibody-positive wells are cloned during ds and Applications by techniques such as the soft agar technique of Kruse and Paterson editing, Academic Press, 1973.

[0041] about to about 4 days after priming primed Balb / c mice with pristane 2x10 ⁶ to about 6x10 ⁶ to about 0.5 per mouse hybridoma cells
The monoclonal antibody is produced in vivo by injecting ml. About 8-12 days after cell introduction, ascites fluid is collected and the monoclonal antibody is purified by techniques known in the art.

DME containing about 2% fetal calf serum to obtain a sufficient amount of specific mAb
In vitro production of the anti-ras carboxyl-terminal processing protein mAb is performed by expanding hybridomas in M.

The antibody titers of ascites fluid or hybridoma cultures were determined by various serological or immunological assays, including sedimentation, passive agglutination, enzyme-linked immunosorbent assay (ELIS).
A) and radioimmunoassay (RIA) techniques.
A similar assay is used to detect the presence of the ras carboxyl-terminal processing protein in body fluids or tissue and cell extracts.

Production of Monospecific Antibodies to Produce Antibodies Specific for a Ras Carboxyl-Terminal Processing Protein Polypeptide Fragment or a Full-Length Nascent Ras Carboxyl-Terminal Processing Protein Polypeptide or Individual Ras Carboxyl-Terminal Processing Protein Subunits It will be readily apparent to one of ordinary skill in the art that the above described methods for doing so can be utilized. In particular, it is readily apparent to those skilled in the art that monospecific antibodies specific to only one ras carboxyl-terminal processing protein subunit or to a fully functional protein can be generated.

A gel support, Affigel-10 (
A ras carboxyl-terminal processing protein antibody affinity column is prepared by adding the antibody to Biorad). Next, the antibody is bound to the gel by an amide bond with the spacer arm. Next, the remaining activated ester was
Quench with M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any unbound antibodies or extraneous proteins. Next, the column was washed with phosphate buffered saline (pH 7).
. Equilibrate to 3) and slowly pass the cell culture supernatant or cell extract containing the ras carboxyl-terminal processing protein or ras carboxyl-terminal processing protein subunit through the column. Next, the column is washed with phosphate buffered saline until the optical density (A ₂₈₀ ) falls to the background, and then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). Next, the purified ras carboxyl end processing protein is dialyzed against phosphate buffered saline.

D, referred to as pHRP, encodes a protein that forms a protein capable of cleaving the terminal 3 amino acids of the ras protein when expressed in bacterial cells
Identify NA clones. Expression of the ras carboxyl-terminal processing protein DNA results in the reconstitution of properties seen in oocytes injected with poly (A) ⁺ RNA encoding the ras carboxyl-terminal processing protein. Activation
The as protein is associated with a number of malignant human tumors, including the breast, liver and colon.
For ras to exert its function, it must be localized to the cell membrane. The mechanism by which this localization occurs is by three different modifications to the immature protein: the addition of lipid component groups, the removal of COOH-terminal amino acids, and the addition of a methyl group to carboxyphenylcysteine. Agents that prevent the removal of carboxyl 3 amino acids are r
It should result in abnormal localization leading to loss of as function.

The present invention also relates to a method for screening a compound that regulates the expression of DNA or RNA encoding a ras carboxyl terminal processing protein and the function of the ras carboxyl terminal processing protein in vivo. The compound that modulates these activities may be DNA, RNA, peptide, protein or an organic molecule other than protein. Compounds can be modulated by expression of DNA or RNA encoding the ras carboxyl-terminal processing protein or by increasing or decreasing the function of the ras carboxyl-terminal processing protein. DNA or R encoding ras carboxyl-terminal processing protein
Compounds that modulate the expression of NA or the function of the ras carboxyl-terminal processing protein can be detected by various assays. The assay may be a simple "yes / no" assay to determine if there is a change in expression or function. The assay can be made quantitative by comparing the level of expression or function of the reference sample with the expression or function of the test sample. Modulators identified in this way are useful as therapeutics.

A kit containing the ras carboxyl-terminal processing protein DNA or RNA, an antibody against the ras carboxyl-terminal processing protein or the ras carboxyl-terminal processing protein can be produced. Such a kit is used to detect DNA that hybridizes to the ras carboxyl-terminal processing protein DNA or to detect the presence of the ras carboxyl-terminal processing protein or peptide fragment in the sample. Such characterization is useful for a variety of purposes, including but not limited to judicial analysis, diagnostic applications, and epidemiological studies.

For screening and measuring the level of ras carboxyl-terminal processing protein DNA, ras carboxyl-terminal processing protein RNA or ras carboxyl-terminal processing protein, the DNA molecule, RNA molecule of the present invention,
Recombinant proteins and antibodies can be used. Recombinant proteins, DNA molecules,
RNA molecules and antibodies serve to form suitable kits for the detection and typing of ras carboxyl-terminal processing protein activity or expression in tissue samples. Such a kit comprises a compartmentalized carrier suitable for holding in at least one closed container. The carrier may further comprise a reagent such as a recombinant ras carboxyl-terminal processing protein or an anti-ras carboxyl-terminal processing protein antibody suitable for detecting the ras carboxyl-terminal processing protein. The carrier may also include means for detection, such as a labeled antigen or enzyme substrate.

[0050] A nucleotide sequence complementary to the DNA sequence encoding the ras carboxyl-terminal processing protein can be synthesized for antisense therapy. These antisense molecules include DNA, stable derivatives of DNA such as monothiophosphate or methylphosphonate, RNA, 2'-O-alkyl RN
A stable derivative of RNA, such as A, or other ras carboxyl-terminal processing protein antisense oligonucleotide mimic. Microinjection,
The ras carboxyl-terminal processing protein antisense molecule can be introduced into cells by liposome encapsulation or by expression from a vector bearing the antisense sequence. Ras carboxyl-terminal processing protein antisense therapy may be particularly useful for the treatment of diseases where reducing ras activity responsible for tumorigenesis is beneficial.

According to known methods, such as by admixing a pharmaceutically acceptable carrier, ras carboxyl-terminal processing protein DNA, ras carboxyl-terminal processing protein RNA or ras carboxyl-terminal processing protein or ras
A pharmaceutically useful composition comprising a modulator of carboxyl-terminal processing protein activity can be formulated. Examples of such carriers and methods of compounding can be found in Remington's Pharmaceutical Sciences. Such compositions contain an effective amount of protein, DNA, RNA or modulator to form a pharmaceutically acceptable composition suitable for effective administration.

[0052] The therapeutic or diagnostic composition of the present invention is administered to an individual in an amount sufficient to treat or diagnose a disease characterized by altered ras carboxyl terminal processing activity. The effective amount can vary depending on various factors such as the condition, weight, sex and age of the individual. Other factors include the mode of administration. The pharmaceutical composition can be given to an individual by various routes, such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that contains additional chemical moieties that are not normally part of the base molecule. Such components can improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, those components can reduce the undesirable side effects of the base molecule or reduce the toxicity of the base molecule. Examples of such ingredients are Remington's Pharmaceutical Scie
nces.

[0054] Disclosed herein are the appropriate dosages specified by routine testing to obtain optimal inhibition of the ras carboxyl-terminal processing protein or its activity while minimizing any possible toxicity. The compound identified according to the method can be used alone. In addition, co-administration or sequential administration of other agents may be desirable.

The present invention also has the object of providing topical, oral, systemic and parenteral formulations suitable for use in the novel treatment methods of the present invention. A composition containing a compound or modulator identified according to the present invention as an active ingredient for use in modulating ras carboxyl terminal processing protein can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. . For example, compounds or modulators may be administered as tablets, capsules (including time release and sustained release formulations, respectively), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions. It can be administered in a suitable oral dosage form or by injection. Similarly, they can be administered intravenously (both bolus and infusion), intraperitoneally, subcutaneously, topically with or without occlusion, or intramuscularly, all using forms well known to those skilled in the pharmaceutical arts. It may be administered. An effective, but non-toxic, amount of the desired compound can be used as a ras carboxyl-terminal processing protein modulator.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient per day. For oral administration, preferably 0.01, 0.05, 0.1, 0.5, to adjust the dosage to the patient to be treated, depending on the condition.
Provides the composition in the form of a scored or unscored tablet containing 1.0, 2.5, 5.0, 10.0, 15.0, 25.0 and 50.0 milligrams of active ingredient. . Usually, from about 0.0001 mg / kg body weight to about 100 mg / day
It provides an effective amount of drug at dosage levels up to kg body weight. More specifically, the range is from about 0.001 mg / kg body weight to 10 mg / kg body weight per day. Adjust the dosage of the ras carboxyl-terminal processing protein modulator when combined to obtain the appropriate effect. On the other hand, the dosages of these various drugs can be independently optimized and combined in order to obtain synergistic results with less pathological conditions than using either drug alone.

Conveniently, the compounds or modulators of the present invention may be administered in a single daily dose, or the entire daily dose may be administered in two, three or four divided doses daily. In addition, the compounds or modulators of the present invention can be administered in intranasal form by topical use of a suitable intranasal vehicle or by the transdermal route using transdermal skin patches well known to those skilled in the art. For administration in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

The active agents can be administered simultaneously for combination treatment with more than one active agent, where the active agents are separate dosage formulations, or they can each be administered separately It can be administered at staggered times.

The dosage regimen utilizing the compounds or modulators of the present invention may include the type, type, age, weight, sex, and medical condition of the patient; the severity of the disease to be treated; the route of administration; The choice is made according to various factors, including the particular compound used. A physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the disease. The highest accuracy in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of drug efficacy at the target site. This involves taking into account the distribution, equilibrium and elimination of the drug.

In the methods of the present invention, the compounds or modulators described in detail herein form the active ingredient and are intended for the intended dosage form, ie, oral tablet, capsule, elixir, syrup, etc. Typically administered in admixture with a suitable pharmaceutical diluent, excipient or carrier, appropriately selected and consistent with common pharmaceutical practice, collectively referred to herein as "carrier" materials. Is done.

For example, for oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient is combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. it can. In addition, when appropriate or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be included in the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose. Including rolls, polyethylene glycol, wax, etc. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

In liquid form, the active pharmaceutical ingredient can be combined with a suitably flavored suspending or dispersing agent such as synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. Other dispersants that can be used include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. When intravenous administration is desired, isotonic formulations generally containing a suitable preservative are employed.

Topical formulations containing the active pharmaceutical ingredient can be prepared using various well-known in the art such as, for example, alcohol, aloe veragel, allantoin, glycerin, vitamin A and E oils, mineral oil, myristyl PPG propionate, and the like. It can be mixed with carrier materials to form cream or gel formulations, for example, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions and shampoos.

The compounds or modulators of the invention may also be administered in the form of liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from various phospholipids such as cholesterol, stearylamine or phosphatidylcholine.

The compounds of the present invention may also be delivered by using monoclonal antibodies as individual carriers linking the compound molecules. The compounds or modulators of the present invention may also be linked to suitable polymers as targetable drug carriers. Such polymers are polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropyl methacryl-amide phenol, polyhydroxy-ethyl aspartamide phenol (polyhydroxy-ethylaspartamide).
(ephenol), or polyethylene oxide polylysine substituted with palmitoyl residues. In addition, a class of biodegradable polymers useful for achieving controlled release of a compound or modulator of the present invention, such as polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters,
It can be linked to crosslinked or amphiphilic block copolymers of polyacetals, polydihydro-pyrans, polycyanoacrylates and hydrogels.

For oral administration, the compounds or modulators can be administered in capsule, tablet or bolus form, or they can be incorporated into animal feed. Capsules, tablets and boluses comprise the active ingredient in combination with a suitable carrier vehicle such as starch, talc, magnesium stearate or dicalcium phosphate. These unit dosage forms are prepared by thoroughly mixing the active ingredient with the appropriate finely divided inert ingredients, including diluents, extenders, disintegrants and / or binders, to give a homogeneous mixture. I do. Inactive ingredients do not react with the compound or modulator and are non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils and the like. These preparations can contain an amount of active and inactive ingredient which is widely varied by a number of factors such as the size and type of animal species to be treated and the type and severity of the infection. Also, by simply mixing the compound with the feed,
Alternatively, the active ingredient may be administered as an additive to the feed by adding the compound to the surface of the feed. Alternatively, the active ingredient can be mixed with an inert carrier,
The resulting composition can then be mixed with the feed or given directly to the animal. Suitable inert carriers are coarse corn flour and citrus coarse flour (citrus).
meal), fermentation residues, soya grits, dried cereals and the like. The active ingredient may be combined with these inert carriers by grinding, stirring, milling or rolling so that the final composition contains 0.001 to 5% by weight of active ingredient. Mix.

Alternatively, the compounds or modulators can be administered parenterally by injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Infusion may be intramuscular, intraluminal, intratracheal or subcutaneous. Injectable preparations consist of the active ingredient in admixture with a suitable inert liquid carrier.
Acceptable liquid carriers include vegetable oils such as peanut oil, cottonseed oil, sesame oil and the like, and organic solvents such as solketal, glycerol formal and the like. Alternatively, an aqueous parenteral formulation may be used. Vegetable oil is a preferred liquid carrier. Formulations are prepared by dissolving or suspending the active ingredient in a liquid carrier so that the final formulation will contain from 0.005 to 10% by weight of the active ingredient.

Topical use of the compounds or modulators is possible by using a liquid drench or shampoo containing the compounds or modulators of the invention as an aqueous solution or suspension. These formulations generally contain a suspending agent such as bentonite, and usually also contain an antifoaming agent. Formulations containing from 0.005 to 10% by weight of active ingredient are acceptable. Preferred formulations are those containing from 0.01 to 5% by weight of a compound or modulator of the present invention.

The following examples illustrate the invention, however, the invention is not limited thereto.

[0070]

EXAMPLES Example 1 RNA Blot Clontech Laboratories, Inc. , Palo Alto
, CA purchased two Northern blots. Human Multiple Tissue Northern (Hum
An Multiple Tissue Northern (MTN ^™ ) blot (Cat. No. 7760-1) was run on eight different human tissues run on a denaturing formaldehyde 1.2% agarose gel and transferred to a charge-modified nylon membrane. Contained approximately 2 μg of poly A ⁺ RNA per lane. Lanes 1-8 contain in turn RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. A human cancer cell line multiple tissue Northern (Human Cancer Cell Line Multiple Tissue Northern (MTN ^™ ) ™) blot (catalog number 7757-1) contains 1.2% denatured formaldehyde.
It contained approximately 2 μg of poly A ⁺ RNA per lane from eight different human cell lines, run on an agarose gel and transferred to a charge-modified nylon membrane. Lanes 1-8 are, in order, human promyelocytic leukemia HL-60, HeLa (HeLa) cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colon adenocarcinoma SW480, lung cancer RNA from A549 and melanoma G361
It contains. Hybridization conditions Using the Rediprime DNA labeling system (Amersham, catalog number RPN1633) according to the protocol, the probe was subjected to α- ³² P-dC.
Labeled with TP (Amersham, catalog number AA0005). Twenty-five ng of pHRP cDNA (a 928 bp PCR fragment corresponding to the coding region of HRP) was labeled as described above and Clontech Multiple Tissue Northern (MT
^NTM ) Blots were hybridized according to the protocol in the user manual. The blot is stripped according to the protocol described above, and 2
Detection was performed again with 5 ng of β-actin cDNA control probe. The blot was incubated with the probe overnight according to the protocol described above and washed. The blots were exposed to film for different times and developed on a film processor. Blots were also scanned on a Packard Instant Imager. Cloning of HRP A search of the Incyte database using the 916 amino acid sequence of the yeast RCE1 protein identified several hits, including clone # 1786569. Many clones belonged to cluster # 40377. The clusters were combined to form an 890 bp contig called 1786569. Yeast RCE1
And 1786569 contig sequence alignment showed 49.8% nucleotide identity (Gap analysis on GCG). Based on the two nucleotide and amino acid alignments, the 1786569 contig was not a full-length sequence.
Based on the yeast RCE1 sequence, it lacked about 500 bp at the 5 'end. The first 404 bp of the contig corresponds to the last 404 bp of the yeast RCE1 gene. The rest of the contig (486 bp) is the 3 'untranslated sequence. Five reverse-strand PCR primers were designed for the 1786569 contig. λgt10 human colon adenocarcinoma 5 ′ extended cDNA library (catalog number HL3014a) and λgt10 5 ′ and 3 ′ amplimers were obtained from Clontech. Using these amplimers together with the five reverse strand primers for the 1786569 contig, a 1400 bp region (putative HRP
Several fragments (from the beginning of the gene to the end of the 3 'untranslated sequence) were amplified. These fragments were gel isolated using the Geneclean II kit (Bio 101, Inc.), filled in (large fragment of DNA polymerase, BRL), and kinased (T4 DNA kinase, BRL). Next, these products were linked as follows. The coding region of the putative HRP clone had 41% identity in nucleotide sequence and 30% identity in amino acid sequence (Gap analysis in GCG).

Two rapid amplifications of the cDNA end (RACE) were performed using 2 μg of human heart polyA mRNA as starting material. CDNA was synthesized using a Superscript cDNA synthesis kit (Life Technologies). After first strand synthesis, the cDNA was applied to an S-400 column (Pharmacia) for 2 hours.
The product was purified once, and a series of G was added to the 5 'end using Tdt (Life Technologies). 1 μl of the cDNA having the G ligated to the end was
Sense gene specific oligos (usually 24-30mer, Tm80C) and Kle
It was used as a template for PCR amplification (5'race) using C18 primers with ntaq DNA polymerase (35 cycles). This first PCR product
1 μl of the DNA for the second PCR (under the same conditions) with the inner gene-specific oligo
Was. These PCR products were TA cloned (Invitrogen) and
Quenched. Partially deleted due to a very GC rich region upstream of the ORF
Clones were obtained. Next, another round using prostate mRNA as starting material
Marathon kit (Clontech) to perform 5 'race
Using. The kit was used according to the manufacturer's instructions.Plasmid preparation Plasmid PCR-blunt was replaced with Zero-Blunt^TM(Trademark) PCR black
Invitro as a part of the cleaning kit (catalog number K2700-20)
gen. From PCR products purified from nested PCR reactions
Use it as a cloning vector to insert the 1400 bp fragment
Used first. 25 ng of PCR-blunt vector and about 100 ng of in
The PCR products were ligated using a salt. Gibco / BRL T4 DNA rigger
The ligation reaction is continued at 12 ° C. overnight with 1U and 5X ligase buffer.
I did. Ligation is performed according to the protocol provided with the Zeroblunt kit.
The solution was transformed into Top10 cells. Contains the correct insert by PCR
Colonies were confirmed, and LB medium containing kanamycin (50 μg / μl) was
Propagated in the ground. Qiagen tip-100 Plasmid Maxi D
DNA was isolated from the culture using the NA isolation kit. DNA is ACGT, In
c. (Northbrook, IL). N at 5 'end
Using primers containing deI and a BamHI restriction site at the 3 'end
Only the HRP DNA coding region was amplified again by PCR. The PCR product was recloned into PCR-blunt as described above. PCR-blue
The nt HRP construct was digested with the restriction enzymes NdeI and BamHI (BRL) to release the insert, which was gel isolated as described above. Next, the digested ends
PET digested with the resulting HRP fragment with NdeI and BamHI
 16b (Novagen). The ligation solution was transformed into BL21 (DE3) cells (Novagen) according to the protocol.Example 2 Characterization of pHRP Using standard methods previously described and known in the art,
Xenopus laevis oocytes are prepared and injected. Adult female Xenopus laevis with 0.17% tricaine methanesulfonate
After intoxication, the ovaries were surgically removed and adjusted to pH 7.5 with NaOH (mM): NaCl 82.5, KCl 2, MgCl_Two 1, CaCl_Two 1.8, HEP
Place in a dish consisting of ES 5 (OR-2). Break open the ovarian lobe, rinse several times, and 2 in OR-2 containing 0.2% collagenase (Sigma, type 1A)
Shake gently for 5 hours. When about 50% of the follicular layer has been removed,
Oocytes were selected and adjusted to pH 7.5 with NaOH (mM): NaCl 86
, KCl 2, MgCl_Two 1, CaCl_Two 1.8, HEPES 5, pyruvate N
a Placed in medium (ND-96) consisting of 2.5, theophylline 0.5 and gentamicin 0.1 for 24-48 hours before injection. Oocytes contain 50-70 nl of poly (A ⁺ ) Inject RNA (1 mg / ml) or 50 nl ras carboxyl-terminal processing protein RNA (0.1-100 ng). To control oocytes
Inject 50 nl of water. ras carboxyl-terminal processing protein expression
Oocytes are incubated for 2-10 days in ND-96 before analysis. Incu
The incubation and the collagenase digestion are performed at 18 ° C.

[0072] African Xenopus oocytes injected with the ras carboxyl-terminal processing protein poly (A) ⁺ RNA show activity against the synthetic ras carboxyl-terminal substrate. Example 3 The nucleotide sequence of the primary structure of the ras carboxyl-terminal processing protein, pHRP, revealed a single large open reading frame of about 1013 base pairs (FIG. 1). cDNA is about 6 in pHRP
And 5 'and 3' untranslated extensions of about 331 nucleotides. A 338 amino acid protein with an estimated molecular weight (M _r ) of about 40,000 Da showed the first in-frame methionine as the start codon of the predicted open reading frame (FIG. 2).

The predicted ras carboxyl-terminal processing protein was aligned with nucleotide and protein databases and found to be related to yeast ras convertase (RCE1, Genebank accession Z49260). About 30% of the amino acids of the ras carboxyl-terminal processing protein were identical to RCE1. Although Example 4 E. coli expression ras carboxyl-terminal processing蛋 white matter cDNA Cloning pET series into a vector (Novagen), beginning but not limited to, ras-carboxyl-terminal processing protein expression cassette into E. coli expression vector After transfer, a recombinant ras carboxyl-terminal processing protein was produced in Escherichia coli. The pET vector places the ras carboxyl-terminal processing protein expression under the control of a tightly regulated bacteriophage T7 promoter. After introduction of this construct into an Escherichia coli host containing a chromosomal copy of the T7 RNA polymerase gene driven by an inducible lac promoter, the addition of an appropriate lac substrate (IPTG) to the culture results in ra
Expression of the s-carboxyl-terminal processing protein is induced. The level of expressed ras carboxyl-terminal processing protein is determined by the assays described herein.

The cDNA encoding the entire open reading frame of the ras carboxyl-terminal processing protein is inserted into the Ndel / BamHI site of pET16b. Positively oriented constructs are identified by sequence analysis and used to transform expression host strain BL21. The transformant is then used to inoculate a culture for production of the ras carboxyl-terminal processing protein. Cultures can be grown in LB media, the formulation of which is known to those skilled in the art. After growth to OD ₆₀₀ = 1.5, expression of the ras carboxyl-terminal processing protein is induced with 1 mM IPTG at 37 ° C. for 3 hours. Example 5 Cloning of the Ras Carboxyl-Terminal Processing Protein cDNA into a Mammalian Expression Vector Cloning the coding sequence of HRP (encoding 338 amino acids, nt 6-1019) using the ecdysone-inducible expression system (Invitrogen), Was expressed. KpnI of expression vector pIND (Sp1) / V5-HisA
And the coding sequence of HRP cloned into the EcoRI site stably transfected the mammalian cell lines EcR-3T3 and EcR-293 (stably transfected with the regulator vector pVgRXR) under the selection of neomycin.

The transfected cells were treated with 5 μM ponasterone.
) Expression was induced by treatment with A. Untransfected cells were also treated with ponasterone A as a negative control. After 24 hours, the cells were washed with cold PBS and washed with HNTG lysis buffer (50 mM HEPES, pH 7.5, 150 m
M NaCl, 1% Triton X-100, 8% glycerol, 1.5 mM
MgCl ₂ , 2 mM EDTA). 14,000 rpm cell lysate
and spun for 10 minutes and the supernatant and pellet were collected. The protein concentration was determined for each supernatant and 50 μg of protein and solubilized pellet was
Analyzed on a Tris / glycine / SDS gel. Western blots were performed and blots were detected with an anti-6X His antibody (Invitrogen) and an anti-peptide antibody raised against the C-terminus of the protein. Untransfected cells are 6X
No expression of His was shown. Some transfected clonal isolates were positive for HRP expression by anti-peptide antibodies. These cell lines were analyzed for HRP activity by the two methods described herein.

The ras carboxyl-terminal processing protein cDNA can be cloned into the mammalian expression vector pcDNA3 as a HindIII / BamHI fragment generated by PCR from pHRP. Isolating the recombinant, pcD
Called NA3HRP, it was used to transfect mammalian cells (L- cells) by CaPO _4-DNA precipitation. Stable cell clones were selected by growth in the presence of G418. A single G418 resistant clone was isolated, and was shown to contain the entire ras carboxyl-terminal processing protein gene. Clones containing the ras carboxyl-terminal processing protein cDNA were analyzed for expression using immunological techniques such as immunoprecipitation using antibodies specific for the ras carboxyl-terminal processing protein, Western blot and immunofluorescence. Antibodies are obtained from rabbits inoculated with a peptide synthesized from the amino acid sequence predicted from the ras carboxyl-terminal processing protein sequence.

The expression of the ras carboxyl-terminal processing protein and the ras processing activity are tested using cells stably or transiently expressing the ras carboxyl-terminal processing protein. These cells are used to identify and test other compounds for their ability to inhibit the ras carboxyl-terminal processing protein.

Ligating the cassette containing the ras carboxyl-terminal processing protein cDNA in the positive direction with respect to the promoter, to an appropriate restriction site 3 ′ of the promoter;
Identify by restriction site mapping and / or sequencing. These cDNA expression vectors can be transformed into fibroblast host cells, eg, COS-7 (ATCC # CRL1651), by standard methods including but not limited to electroporation or chemical methods (cationic liposomes, DEAE dextran, calcium phosphate).
) And CV-1 tat [Sackevitz et al., Science 238: 15.
75 (1987)], 293, L (ATCC # CRL6362).
Transfected cells and cell culture supernatant can be harvested and analyzed for ras carboxyl-terminal processing protein expression as described herein.

All of the vectors used for mammalian transient expression can be used to establish stable cell lines that express the ras carboxyl-terminal processing protein. It is expected that the unmodified ras carboxyl-terminal processing protein cDNA construct cloned into the expression vector will cause the host cell to make the ras carboxyl-terminal processing protein.

G418, aminoglycoside phosphotransferase; hygromycin,
Hygromycin-B phosphotransferase; stably transfected by co-transfection of any vector containing the ras carboxyl-terminal processing protein cDNA with a drug selection plasmid, including but not limited to APRT, xanthine-guanine phosphoribosyl-transferase Clones can be selected. The level of ras carboxyl-terminal processing protein is quantified by the assays described herein.

The ras carboxyl-terminal processing protein cDNA construct is also ligated into a vector containing an amplifiable drug resistance marker for the production of a mammalian cell clone that synthesizes the highest possible level of the ras carboxyl-terminal processing protein. . After introduction of these constructs into cells, clones containing the plasmid are selected with the appropriate drug, and selection on increasing amounts of drug achieves isolation of overexpressing clones with high copy number plasmid. .

[0082] Full-length ras carboxyl-terminal processing protein cDN into mammalian host cells
Transfection of A results in expression of the recombinant ras carboxyl-terminal processing protein. S baculovirus expression ras Cal Bokishiru terminal processing protein derived from the genome of the baculovirus vector insect cell cloning AcNPV virus cDNA into a vector for expression in Example 6 Insect Cells
Designed to provide high level expression of cDNA in f9 line (ATCC CRL # 1711). Recombinant baculovirus expressing the ras carboxyl-terminal processing protein cDNA is prepared by the following standard method (InVitrogen Maxbac manual): The ras carboxyl-terminal processing protein cDNA construct is constructed using the pAC360 and BlueBac vectors (InVit Vector).
Logen) and various other baculovirus transfer vectors into the polyhedrin gene. Baculovirus transfer vector and linearized AcNP
V genomic DNA [Kitts, P .; A. , Nuc. Acid. Res. 18: 5
667 (1990)] into Sf9 cells, followed by homologous recombination to produce a recombinant baculovirus. Recombinant pAC360
The virus was identified by the absence of inclusion bodies in infected cells, and the recombinant pBl
ueBac virus is identified based on β-galactosidase expression (Summ
ers, M.S. D. And Smith, G .; E. FIG. , Texas Agriculture Exp. Station Bulletin No. 1555). After plaque purification, ras carboxyl-terminal processing protein expression is measured by the assays described herein.

The cDNA encoding the entire open reading frame of the ras carboxyl-terminal processing protein is inserted into the BamHI site of pBlueBacII. Positively oriented constructs were identified by sequence analysis and the linear AcNPV mild (mild)
) Used to transfect Sf9 cells in the presence of type DNA.

[0084] A true, active ras carboxyl-terminal processing protein is found in the cytoplasm of infected cells. Active ras carboxyl-terminal processing protein is extracted from infected cells by hypotonic or solvent lysis. Example 7 Cloning of ras Carboxyl-Terminal Processing Protein cDNA into Yeast Expression Vector Recombinant ras carboxyl-terminal processing protein after inserting a cistron into an expression vector designed to direct intracellular or extracellular expression of a heterologous protein Is produced in the yeast Saccharomyces cerevisiae. In the case of intracellular expression, a vector such as EmBLyex4 is linked to cistron by "Rinau
, U.S. Et al., Biotechnology 8: 543-545 (1990); J. et al. Biol. Chem. 265: 4189-419 2 (1989). The extracellular expression, linking the ras carboxyl-terminal processing protein cistron yeast expression vector to give a ras carboxyl-terminal processing protein NH ₂ terminus secretion signal (a yeast or mammalian peptide) [Jacobson, M. A. Riet L., Gene 85: 511-516 (1989); And Bellon N.M. Biochem. 28: 2941-2
949 (1989)].

These vectors provide a pAVE1> 6 [Steep O.D. that attaches a human serum albumin signal to the expressed cDNA. Biotechnology 8: 42-4
6 (1990)] and the vector pL8PL [Yamamoto, Y .; Biochem. 28: 2728-
2732]. Furthermore, the vector pVEP [Ec
ker, D .; J. J. Biol. Chem. 264: 7715-7719 (1
989), Sabin, E. et al. A. BioTechnology 7: 705-709 (1989); McDonnell D .; P. , Mol. Cell Bio
l. 9: 5517-5523 (1989)] to express a ras carboxyl-terminal processing protein as a fusion protein bound to ubiquitin in yeast. The level of expressed ras carboxyl-terminal processing protein is determined by the assays described herein. Example 8 Purification of Recombinant Ras Carboxyl-Terminal Processing Protein Recombinantly produced ras carboxyl-terminal processing protein can be purified by antibody affinity chromatography.

Gel support, Affigel, which is preactivated with N-hydroxysuccinimide ester so that the antibody forms a covalent bond with the agarose gel bead support
A ras carboxyl-terminal processing protein antibody affinity column is prepared by adding anti-ras carboxyl-terminal processing protein antibody to -10 (Biorad). Next, the antibody is bound to the gel by an amide bond with the spacer arm. The remaining activated ester is then quenched with 1 M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any unbound antibodies or irrelevant proteins. The column is then equilibrated in phosphate buffered saline (pH 7.3) with a suitable membrane solubilizing agent, such as a solvent, and the cell culture supernatant containing the solubilized ras carboxyl-terminal processing protein or Slowly pass the cell extract through the column. The column is then washed with phosphate buffered saline with solvent until the optical density (A ₂₈₀ ) falls to the background, and then 0.23 M glycine-
Elute the protein with HCl (pH 2.6). Next, the purified ras carboxyl terminal processing protein is dialyzed against phosphate buffered saline. The recombinantly produced ras carboxyl terminal processing protein is also purified by affinity chromatography. The ras carboxyl-terminal processing protein is expressed as a tandem fusion protein with a purification tag including but not limited to glutathione S-transferase (GST) and polyhistidine residues (His6). The cells or bacteria that express the protein are harvested and lysed chemically and / or by mechanical means. The lysate is added to the pH-adjusted slurry of the GST and His6 fusion protein affinity substrates, glutathione sepharose and Ni ²⁺ charged agarose, respectively. Wash the affinity substrate thoroughly and elute the protein with glutathione or imidazole for the GST and His6 fusion proteins respectively. Expression is determined by Western blot analysis using an antibody against the fusion tag or against the ras carboxyl-terminal processing protein. FIG. 6 is a diagram of the plasmid used to produce the human ras carboxyl-terminal protease-His6 fusion protein. Example 9 Method for Detecting Human Ras Protease Activity Protease activity is measured in microsomal preparations made from transfected cell lines. The method of preparation is described in Methods in Enzymlog.
y Adapted from Volume 96, 88-89. Briefly, cells are collected and thoroughly homogenized with a Dounce homogenizer (Type B pestle). The homogenate is centrifuged at 1000 g _av for 10 minutes. Collect the supernatant, layer on a sucrose cushion, Beckm
30 at 140,000 g _av in an TL ultracentrifuge (TLA 100.3 rotor)
Subject to centrifugation for minutes. The resulting cell pellet is 250 mM sucrose, 50 mM
Resuspend in buffer containing triethanolamine pH 7.5 and 1 mM DTT. Perform protein measurements and store microsomes at -70 ° C. Fluorescence resonance energy transfer (FRET) assay A peptide substrate corresponding to the C-terminal of K-rasB with a fluorophore and a chromophore group attached to either end [peptide sequence: (Aedens) ES
KTK (farnesyl) CVIM (Dabcyl) K-amide] was used. NIH3T3 HR in a buffer containing 25 μM peptide in 200 mM Hepes pH 7.4, 100 mM NaCl and 5 mM MgCl _2.
The cells were incubated with microsomal fractions (10 μg) made from P-transfected cells (clone 5) and non-transfected NIH3T3 cells. Reaction 3
Excitation at 50 nm and emission spectra are recorded on a LS50B spectrofluorometer (Perkin Elmer) between 400-500 nm at 5 minute intervals for 1 hour. Additional spectra were recorded at 24 hours. Transfected cells show a time-dependent cleavage of the substrate and non-transfected cells show minimal activity (FIG. 4). High Performance Liquid Chromatography (HPLC) Analysis A peptide substrate [peptide sequence: WKKKKSKTK (farnesyl) CVIM] corresponding to the C-terminal of K-ras having N-terminal trp was used. 500 μM
Were incubated with microsomes (4-20 μg of total protein) in assay buffer (200 mM Hepes pH 7.4, 100 mM NaCl, 5 mM MgCl ₂ ) at 30 ° C. for 24 hours. The protein was precipitated and the isolated peptide was run by HPLC to determine the extent of cleavage. 100% TFA in H ₂ O (mobile phase A) to 0.07% TFA in acetonitrile (mobile phase B).
% A for 10 minutes followed by a linear gradient from 0-100% B in 50 minutes, 5% in 100% B
A Water 996 photodiode array (Photodiode Array) detector was run using a Vydac protein-peptide C18 column (part number 218TP54) using a gradient consisting of 100-0% B in 5 minutes and 100% A in 5 minutes. Separation was performed on a Waters 625 HPLC equipped. 50% of 2 volumes freshly prepared daily from a stock of 4% zinc sulfate and pure acetonitrile
The protein was precipitated by adding 2% zinc sulfate in acetonitrile. The mixture was centrifuged for 5 minutes in a microcentrifuge and the supernatant was injected into the HPLC system above, which had been equilibrated to 100% A. Transfected cells show the appearance of deleted peptide substrate eluting from the column consistent with known deleted peptide standards. Enzymatically produced product and synthetic ras protease product peptide [WKKKKSKT
K (farnesyl) C] and LC-MS confirmation that the enzymatically generated product has a mass consistent with the expected cleavage product of the substrate peptide, the approximately 30% observed in the rp-HPLC analysis. Product identity was confirmed.
Thus, about 181 by cleavage of the C-terminal peptide bond of the (farnesyl) -C residue
The conversion of 1 amu substrate to about 1467 amu product is shown (FIG. 5).

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the human ras carboxyl-terminal processing protein.

FIG. 2 shows the amino acid sequence of the human ras carboxyl-terminal processing protein.

FIG. 3. Northern blot analysis of human ras carboxyl-terminal processing protein expression.

FIG. 4 shows the activity of a recombinantly produced human ras carboxyl-terminal processing protein.

FIG. 5 shows the identification of the active recombinant human ras carboxyl-terminal processing protein enzyme product by HPLC.

FIG. 6: DNA encoding the recombinant human ras carboxyl-terminal processing protein
Shows a map of a plasmid containing

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C12N 1/19 4C085 C07K 16/40 1/21 4H045 C12N 1/19 9/50 1 / 21 C12P 21/02 C 5/10 21/08 9/50 C12N 15/00 ZNAA C12P 21/02 A61K 37/02 21/08 C12N 5/00 B (81) Designated countries EP (AT, BE, CH, CY , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, D, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Jillsonson, Dana 1972 Pennsylvania, U.S.A., Upper Lonely Cottage Road 1343 (72) Inventor Patel, Reka 08807, New Jersey, United States 08807 Bridgewater Cabot Hillroad 572 (72) Inventor Jyorif, Linda 08502 Bellmead Davenport Way, New Jersey, USA 16 (72) Inventor Galind, Jyoze 92173, San Diego, Monterey Pine Drive, Calif., USA 1550-Bee (72) Inventor Huber, Arne 92071, San Dempster Drive, Calif., United States 9434 F Term (Reference) 4B024 AA01 AA12 BA07 BA14 BA44 CA04 DA02 DA06 DA12 HA12 4B050 CC03 DD11 4B064 AG01 CA02 CA06 CA10 CA19 DA01 DA13 4B065 AA26X AA72X AA90X AA93Y AB01 BA02 CA33 CA44 CA46 4C084 AA02 CA01 BA23 BA01 BA01 BA23 ZC202 ZC422 4C085 AA13 AA14 BB11 CC01 CC04 DD62 DD86 DD88 4H045 AA10 AA20 AA30 BA10 CA50 DA89 EA28 FA74

Claims (23)

[Claims] 1. An isolated and purified DNA molecule encoding a ras carboxyl-terminal processing protein, wherein said protein optionally functions as a ras processing enzyme or a functional derivative thereof. 2. The isolated and purified DNA molecule of claim 1 having a nucleotide sequence selected from the group consisting of (SEQ ID NO: 1) and functional derivatives thereof. 3. The isolated of claim 1, wherein said DNA molecule is genomic DNA.
A purified DNA molecule. 4. An expression vector for expressing a ras carboxyl-terminal processing protein in a recombinant host, said vector comprising a recombinant gene encoding a ras carboxyl-terminal processing protein, said protein comprising a ras processing enzyme and its function. Expression vector that optionally functions as a functional derivative. 5. An expression vector comprising a cloned gene encoding a ras carboxyl-terminal processing protein having a nucleotide sequence selected from the group consisting of (SEQ ID NO: 1) and functional derivatives thereof, said protein comprising: 5. The expression vector of claim 4, wherein optionally functions as a ras processing enzyme. 6. The expression vector of claim 4, wherein said expression vector comprises genomic DNA encoding a ras carboxyl terminal processing protein, said protein optionally functioning as a ras processing enzyme. 7. A recombinant host cell containing a recombinantly cloned gene encoding a ras carboxyl-terminal processing protein, wherein the protein optionally functions as a ras processing enzyme or a functional derivative thereof. . 8. The recombinant host cell of claim 7, wherein said gene has a nucleotide sequence selected from the group consisting of (SEQ ID NO: 1) and functional derivatives thereof. 9. The recombinant host cell of claim 7, wherein said cloned gene encoding a ras carboxyl-terminal processing protein is genomic DNA. 10. A protein in a substantially pure form that functions as a ras carboxyl-terminal processing protein, wherein the protein optionally functions as a ras processing enzyme. 11. The protein according to claim 10, which has an amino acid sequence selected from the group consisting of (SEQ ID NO: 2) and a functional derivative thereof. 12. A monospecific antibody that immunologically reacts with a ras carboxyl-terminal processing protein, said protein optionally functioning as a ras processing enzyme. 13. The antibody of claim 12, wherein the antibody interferes with the activity of the ras carboxyl-terminal processing protein. 14. A method for expressing a ras carboxyl-terminal processing protein, said protein optionally functioning as a ras processing enzyme in a recombinant host cell, and (a) introducing the expression vector of claim 4 into a suitable host cell. And (b) culturing the host cell of step (a) under conditions that allow expression of the ras carboxyl-terminal processing protein from the expression vector. 15. A method for identifying a compound that modulates the activity of a ras carboxyl-terminal processing protein, comprising: (a) combining a ras carboxyl-terminal processing protein with a modulator of the ras carboxyl-terminal processing protein activity;
Optionally functioning as a processing enzyme; and (b) measuring the effect of the modulator on the protein. 16. The method of claim 15, wherein the effect of the modulator on the protein is inhibition of ras carboxyl-terminal processing protein activity. 17. The method of claim 15, wherein the effect of the modulator on the protein is inhibition of ras function. 18. The method of claim 17, wherein ras localization is prevented from the cell membrane. 19. The compound of claim 1, wherein said compound is a modulator of ras function.
A compound that is active in the method of 5. 20. The compound active in the method of claim 15, wherein said compound is an antagonist of ras carboxyl terminal processing protein or ras protein. 21. The compound active in the method of claim 15, wherein said compound is a modulator of ras carboxyl-terminal processing protein expression. 22. A pharmaceutical composition comprising a compound active in the method of claim 15, wherein said compound is a modulator of ras carboxyl-terminal processing protein activity. 23. A method of treating a patient in need of such a treatment for a disease caused by an activated ras protein, comprising administering an active ras carboxyl terminal processing protein modulating compound in the method of claim 15.