WO1993004186A1

WO1993004186A1 - B-raf protein kinase

Info

Publication number: WO1993004186A1
Application number: PCT/US1992/006845
Authority: WO
Inventors: Ulf R. Rapp; Stephen D. Showalter
Original assignee: United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1991-08-13
Filing date: 1992-08-13
Publication date: 1993-03-04
Also published as: AU2507092A

Abstract

The present invention relates, in general, to a B-raf protein kinase. In particular, the present invention relates to a DNA segment encoding a B-raf protein kinase; a polypeptide encoded by the DNA segment; antibodies to the protein kinase; kits containing the antibodies; a recombinant DNA molecule containing the DNA segment; a cell containing the recombinant DNA molecule; and a method of producing a polypeptide encoded by the DNA segment.

Description

B-RAF PROTEIN KINASE

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of application Serial Number 07/531,950 filed June 1, 1990 the contents of which are incorporated herein by reference.

Field of the Invention

The present invention relates, in general, to a B-raf protein kinase. In particular, the present invention relates to a DNA segment encoding a B-raf protein kinase; a polypeptide encoded by said DNA segment; antibodies to" the polypeptide; a recombinant DNA molecule containing the DNA segment; a cell containing the recombinant DNA molecule; and a method of producing a polypeptide encoded by the DNA segment.

Background Information

Three isotypes of raf protein kinases have been identified to date in man: raf-1 (Bonner, T.I. et al. (1985) Mol. Cell. Biol. 5:1400-1407; Bonner, T.I. et al. (1986) Nucleic Acids Res. 1 :1009-1015), A-raf (Huleihel, M. et al. (1986) Mol. Cell. Biol. 6:2655-2662.; Huebner, K. et al. (1986) Proc. Natl. , Acad. Sci. USA 83:3934-3938; Beck, T.W.- et al. (1987) Nucleic Acids Res. 15:595-609), and B-raf (Ikawa, S. et al. (1988) Mol. Cell. Biol.

8:2651-2654). These serine/threonine specific raf protein kinases (Moelling, K. et al. (1984) Nature 312:558-561; Schultz, A.M. et al. (1988) Oncoσene 2:187-193) function in the transduction of mitogenic signals from the cell membrane to the nucleus (Rapp, U.R. et al. (1988a) The Oncoqene Handbook, Reddy, E.P. et al. (eds.) Elsevier Press: Amsterdam, pp. 213-253; Rapp, U.R. et al. (1988b) Cold Spring Harbor Svmp. Ouan. Biol. 53:173-184, Cold Spring Harbor Press: Cold Spring Harbor, N.Y; Heidecker, G. et al. (1989) Genes and Signal Transduction in Multistage Carcinoqenesis, Colburn, N.H. (ed.), Marcel Dekker, Inc.: New York, pp. 339-373). Evidence for this comes from the observation that in NIH3T3 cells in which ras function is blocked, mitogenic signal flow was also aborted. This block was overcome by activated raf-1 or A- raf consistent with a position of raf in signal transduction downstream of ras (Mulcahy, L.S. et al. (1985) Nature 313:241-243; Smith, M.R. et al . (1986) Nature 320:540-543; Rapp, U.R. et al. (1988a) The Oncoσene Handbook, Reddy, E.P. et al. (eds.) Elsevier Press:

Amsterdam, pp. 213-253). Moreover, in various cell types mitogenic stimulation increases the phosphorylation status as well as the kinase activity of Raf-1 (Morrison, D.K. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859; Morrison, D.K. et al. (1989) Cell 58:649-657; Rapp, U.R. et al. (1991) Oncogene 6:495-500. Furthermore, mitogen treatment of fibroblasts induces the translocation of activated Raf-1 kinase from the cytoplasm to the nucleus (Rapp, U.R. et al. (1988b) Cold Spring Harbor Svmp. Ouan. Biol. 53:173-184, Cold Spring Harbor Press: Cold Spring Harbor, N.Y). While the characterization of Raf-1 has made rapid progress, other raf family members have not been extensively studied. raf-1 , (c-raf) was originally identified as the cellular homologue of v-raf (Rapp, U.R. et al. (1983)

Proc. Natl. Acad. Sci. USA 80:4218-4222) and encodes a 74 Kd protein translated from a message which is ubiquitously expressed in all tissues (Bonner, T.I. et al. (1985) Mol. Cell. Biol. 5:1400-1407; Bonner, T.I. et al. (1986) Nucleic Acids Res. 14:1009-1015; Storm et al. , 1990). Subsequent screening of a cDNA library under less stringent conditions resulted in the isolation of A-raf which encodes a 68 Kd protein and shows a restricted pattern of RNA expression (Huleihel et al., 1986; Beck et al., 1987; Storm, S.M. et al. (1990) Oncogene 5:345-350). The most recently identified member, B-raf, was isolated as a transforming gene in NIH3T3 cell transfection assays with human Ewing sarcoma DNA (Ikawa, S. et al. (1988) Mol. Cell. Biol. 8:2651-2654); B-raf expression is highly restricted with highest levels in cerebrum and testes (Storm, S.M. et al. (1990) Oncoσene 5:345-350). So far, only a partial B-raf sequence corresponding to the kinase portion has been established (Ikawa, S. et al. (1988) Mol. Cell. Biol. 8:2651-2654). The present invention provides a full length human B-raf cDNA from testes as well as the corresponding B-raf protein.

SUMMARY OF THE INVENTION

It is a general object of this invention to provide a DNA segment encoding a B-raf protein kinase.

It is another object of the invention to provide a polypeptide corresponding to a B-raf protein kinase. It is a further object of the invention to provide a recombinant DNA molecule comprising a vector and a DNA segment encoding a B-raf protein kinase.

It is yet another object of the invention to provide a cell that contains the above-described recombinant DNA molecule.

It is a further object of the invention to provide a method of producing a polypeptide having an amino acid sequence corresponding to a B-raf protein kinase. It is another object of the invention to provide a hybridoma which produces a B-raf protein kinase specific monoclonal antibody.

It is a further object of the invention to provide a B-raf protein kinase specific monoclonal antibody.

It is another object of the invention to provide a diagnostic kit comprising at least one of the above- described monoclonal antibodies.

Further objects and advantages of the present invention will be clear from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The nucleotide sequence of human B- raf cDNA and the deduced amino acid sequence of the B-raf protein. Conserved regions (CR) are indicated including the cysteine rich putative 'zinc finger' region (CR1), the serine/threonine rich region (CR2), and the ATP binding region of the kinase domain (CR3). The G—X-G-X-X-G sequence is indicated by asterisks, the conserved lysine by an arrow and the APE sequence by +++ signs. The hexanucleotide polyadenylation signals AATAAA are indicated by boxes.

Figure 2. Detection of the B-raf kinase. (a) time course of induction of a recombinant T7-gene10/B—raf fusion protein. E. coli carrying the expression vector pGemex/B-Raf were induced with IPTG. Crude lysates prepared at various timepoints were analyzed by Western blotting with a polyclonal rabbit antiserum (# 301) raised against the carboxy terminal B-raf peptide. B-raf S12, a monoclonal^' antibody specific for the same peptide and the pan-raf monoclonal antibody URP26K. Uninduced (UI) lysates are shown as controls. (b) detection of endogenous B-raf protein and associated kinase activity in brain. Mouse brain tissue was homogenized in a Dounce homogenizer in TBST buffer (150 mM NaCl, 20 mM Tris HC1, pH 7.5, 1% Triton X-100) supplemented with 2mM EDTA and 1 mM PMSF and centrifuged for 20 min at 20,000g. The supernatant fraction was immunoprecipitated with #301 antiserum in the presence (+) or absence (-) of specific competing peptide antigen. After extensive washes with TBST-buffer, the immunoprecipitate was incubated in kinase buffer (TBST, containing 5 mM MgCl₂, 5 mM MnCl₂) with 10 μCi of [³²P] γ-ATP for 5 min at 25°C and washed again. Samples were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. After autoradiography the membrane was immunostained with a non-radioactive alkaline phosphatase reaction using the mouse monoclonal antibody (B-raf S12) specific for the carboxy-terminus of B-raf.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to B-raf protein kinase. More specifically, the present invention relates to a DNA segment coding for a polypeptide having an amino acid sequence corresponding to a B-raf protein kinase. In one embodiment, the DNA segment has the sequence shown in SEQ ID N0:1, a unique portion thereof (unique portion being defined herein as fragments of at least 15, 20, 30, or 100 nucleotides) or allelic or species variation thereof. In another embodiment, the DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, or allelic or species variation thereof.

In another embodiment, the present invention relates to a polypeptide free of proteins with which it is naturally associated (or bound to a solid support) and having an amino acid sequence corresponding to a B-raf protein kinase, or a unique portion thereof (unique portion being defined herein as being at least 5, 7, 10, or 25 amino acids in length). In one preferred embodiment, the polypeptide has the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or a unique portion of such a sequence.

In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment coding for a polypeptide corresponding to a B-raf protein kinase, as described above. In a preferred embodiment, the encoding segment is present in the vector operably linked to a promoter. In a further embodiment, the present invention relates to a cell containing the above described recombinant DNA molecule. Suitable host cells include procaryotes (such as bacteria, including E_j_ coli) and both lower eucaryotes (for example yeast) and higher eucaryotes (for example, mammalian cells). Introduction of the recombinant molecule into the host cell can be affected using methods known in the art.

In another embodiment, the present invention relates to a method of producing the above-described polypeptide comprising culturing the above-described cells under conditions such that the above-described DNA segment is expressed and the encoded polypeptide thereby produced, and isolating the polypeptide.

In a further embodiment, the present invention relates to B-raf protein kinase specific monoclonal antibody, or binding fragment thereof. Monoclonal antibody B-raf 12S is preferred.

The monoclonal antibodies of the invention are produced by hybridomas, advantageously murine hybridomas, that can be prepared and selected as described in the Examples that follow and as is known in the art. For example, a mouse can be immunized by tail scratch with a recombinant B-raf protein, a booster inoculation can be given, and, after a time sufficient to induce an immune response, the mouse can be sacrificed and the spleen and/or lymph cells are obtained and fused, advantageously, with myeloma cells (preferably, murine cells), using known techniques. The resulting cells, which include the fused hybridomas, can be allowed to grow in a selective medium, such as HAT-medium, and the surviving cells are grown in such medium using limiting dilution conditions. The cells can be grown in a suitable container, e.g., microtitre wells, and the supernatants are screened for monoclonal antibodies having the desired specificity.

The invention also relates to useful binding fragments of the B-raf protein kinase specific monoclonal antibodies. The antibody fragments can be obtained by conventional techniques. For example, useful binding fragments can be prepared by digestion of the antibody using papain or pepsin. B-raf fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens. The receptors and their fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See for example, Microbiology, Hoeber Medical Division (Harper and Row, 1969), Landsteiner,

Specificity of Serological Reactions (Dover Publications, New York, 1962) and Williams et al., Methods in Immunology and Immunochemistry, Vol. 1 (Academic Press, New York, 1967).

While the examples of the antibodies of the invention are of the IgG class and are from a murine source, this is not meant to be a limitation. The specified antibodies and antibodies functionally equivalent thereto (that is, capable of binding to the above-described B-raf protein antigens) whether from a murine source, mammalian source, including human, or other sources, or combinations thereof, are included within the scope of this invention, as are antibodies of other classes such as IgA, IgM, IgE, and the like, including isotypes within such classes. Description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, Basic and Clinical Immunology, (Lange Medical Publications, Los Altos, CA, Fourth edition) and references cited therein, and in particular in Kohler and Milstein in Nature 256:495-497 (1975), which discusses one method of generating monoclonal antibodies.

Various conventional methods exist for isolation and purification of the monoclonal antibodies, so as to free monoclonal antibodies from other proteins and other contaminants (see, for example, Goding, in Monoclonal Antibodies: Principals and Practice, Chapter 4, 1986).

The invention also relates to a diagnostic kit for use in detecting the presence of B-raf protein kinase containing cells in a biological sample, which kit is based, for example, on the method described above. In one embodiment, the diagnostic kit comprises (i) the monoclonal antibody or antibodies (or binding fragment(s) thereof) as defined above, and (ii) a conjugate of a specific binding partner for the monoclonal antibody and a label capable of producing a detectable signal. Reagents, such as ancillary agents, for example, buffering agents and protein stabilizing agents and the like, can also be included. The diagnostic kit can further include, where necessary, other members of the signal producing system, of which system the label is a member, agents for reducing background interference in a test, control reagents,, and apparatus for conducting a test. In another embodiment, the diagnostic kit comprises a conjugate of a monoclonal antibody or antibodies of the invention and a label capable of producing a detectable signal. Ancillary agents as mentioned above can also be present.

The present invention is described in further detail in the following non-limiting examples. EXAMPLE 1 cDNA Cloning and Characterization

A 42mer oligonucleotide unique to the B-raf kinase domain which does not cross react with either A-raf or raf-1 was used to screen a human testes cDNA library. Seven positive clones with insert sizes ranging from 0.75 kb to 2.2 kb were identified. Since the major transcript in mouse testes which hybridizes with the 42mer B-raf specific oligonucleotide probe was known to be around 2.4 kb, the clone lambda 3.1 with the largest insert (2.2 kb) was subcloned and sequenced by the dideoxynucleotide chain termination method (Figure 1 ) . Nucleotide sequence analysis and alignments were performed with the University of Wisconsin Genetics Computer Group software package (Devereux, J. et al. (1984) Nucleic Acids Res. 12:387-

395). This clone encompasses a single large open reading frame. Restriction mapping and partial sequence analysis of two other lambda clones Cl 11 and C151 indicated that they were truncated versions of the B-raf clone 3.1. The 2.2 kb insert of B-raf clone 3.1 contains ah initiation codon at position 126 and a termination codon at 2078 which is followed by an untranslated region of 110 nucleotides and a poly(A) stretch of 40 nucleotides. The site of polyadenylation is preceded by two hexanucleotide polyadenylation signals AATAAA that are located at positions 2122 and 2158. The open reading frame of 1953 nucleotides has the capacity to encode a full length B-raf protein of 651 amino acids with a predicted molecular weight of 72.5 kD.

Sequence analysis of B-raf identifies the presence of all three conserved regions CR1 , CR2 and CR3 typical of raf protein kinases. B-raf shows a 75% homology to A-raf and 65% of raf-1 in the CR1 region, which encompasses the putative Zinc finger region. The serine/threonine rich CR2 region has a 47.6% homology with both A-raf and raf-1. CR3 which corresponds to the kinase domain has a 76.4% homology with A-raf and 79% with raf-1. The first 30-40 amino acids at the amino terminus and the last 20 amino acids at the carboxy terminus differ considerably among the various isotypes. They are, however, highly conserved between species in case of raf—1 and A-raf. The amino terminal region of B-raf shows a 54% homology to raf-1 and 52% homology to A-raf.

The putative ATP binding site including the reactive lysine residue located at the sequence motif V—A— X-K are well conserved in B-raf. However, another lysine residue located three amino acids downstream of the V-A—X- K sequence in raf-1 and A-raf is altered to aspartic acid. Like raf-1 , B-raf retains the APE sequence, a tri-amino acid motif that is essential for transformation by src family oncogenes (Kamps, M.P. et al. (1984) Nature 310:589-592). This sequence is substituted by AAE in A- raf (Beck, T.W. et al. (1987) Nucleic Acids Res. 15:595- 609).

Based on the analyses of the transcripts detected by the B-raf oligonucleotide in 36 mouse tissues, it was reported earlier that B-raf has a very restricted pattern of expression and that alternate size transcripts were observed in different tissues (Storm, S.M. et al. (1990) Oncogene 5:345-350). These results were fully confirmed by using a 1.8 kb EcoRI fragment of the B-raf cDNA as probe. Ten and 13 kb transcripts were observed in cerebrum, fetal brain and placenta. High levels of alternate sized transcripts of 2.6 kb and 4.5 kb were observed in testes in addition to very low levels of 10 and 13 kb, which were prevalent in cerebrum and placenta. EXAMPLE 2 Monoclonal Antibodies to B-raf

To detect and characterize the protein product of the B-raf gene, a panel of monoclonal antibodies specific for a conserved region of the raf kinase domain (Kolch, W. et al. (1990) Oncogene 5:713-720), as well as isozyme specific monoclonal and polyclonal sera that were raised against the C-terminal peptide predicted from the cDNA sequence were employed. The specificity of these antibodies was first tested with a B-raf protein expressed in E. coli. Taking advantage of the unique Apal site near the N-terminus of the predicted B-raf coding sequence an Apal-NotI fragment from the KS3.1 B-raf cDNA subclone was inserted into the procaryotic expression vector pGEMEX 1 under the control of the T7 genelO promoter (Studier, S,W. et al. (1986) J. Mol. Biol. 189:113-122). Transcription from this promoter is strictly dependent on T7 RNA polymerase. In an E. coli strain that has been engineered to harbor an IPTG inducible T7 polymerase gene, high level expression of the recombinant B-raf protein was achieved after IPTG induction. The B-raf fusion protein consists of 260 amino acids derived from the B-raf cDNA, resulting in a calculated MW of 77,000 kD. A time course of induction is shown in Figure 2a. Expression is clearly visible after two hours induction and peaks at four hours. Testing various anti-raf antibodies for reactivity with the B-raf fusion protein revealed that both monoclonal and polyclonal sera produced against the predicted B-raf specific C-terminal peptide detected the recombinant B-raf protein. Two of five monoclonal antisera, termed URP26K, and PBA1 , that map into a conserved region of the raf kinase domain and react with c-raf as well as A-raf proteins (Kolch, W. et al. (1990) Oncogene 5:713-720), recognized B-raf with high affinity. Having established the specificity of the antibody reagents, the B-raf specific sera were chosen to detect B-raf proteins in primary mouse tissues, as both N-terminal as well as C— terminal amino acid sequences are invariably conserved amongst raf isozymes from mammalian species. Consistent with mRNA expression data a 75 kD B-raf specific protein could be precipitated from brain and testis lysates using a monoclonal antiserum specific for the C-terminal peptide of B-raf (B-raf 12S). Polyclonal sera raised against the same peptide immunoprecipitated a 77 kD protein in addition to the 75 kD protein. As the 77 kD band also reacted with the monoclonal antisera B-raf 12S and URP26K, it seems to represent an alternative sized B-raf protein rather than unspecific cross reactivity.

Figure 2b shows a representative Western blot of B-raf from brain lysates along with an immunocomplex kinase assay performed with the same lysate. The predominant bands labelled in the in vitro kinase reaction correspond to a 75 kD autophosphorylation product as well as a 120 kD protein. A co-precipitating 120 kD protein is consistently observed in raf-1 as well as in A-raf immunoprecipitates . Its in vitro phosphorylation state parallels changes in Raf-1 kinase activity (Sithanandam, G. et al. (1989a) pp. 171-175, Molecular Diagnostic of Human Cancer, Cold Spring Harbor Laboratory Press: New York; Kolch, W. et al. (1990) Oncogene 5:713-720; Sigel et al. J. Biol. Chem. 265:18472-18480) and, accordingly, it may constitute a common raf substrate (Sigel et al. (1991) J. Biol. Chem. 265:18472).

* * * * *

All publications mentioned hereinabove are hereby incorporated in their entirety by reference. In particular, Sithanadam, G. et al (1990) Oncogene 5:1775 is hereby incorporated in its entirety by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Rapp, Ulf R.

Showalter, Stephen D.

(ii) TITLE OF INVENTION: B-RAF PROTEIN KINASE

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CUSHMAN,DARBY & CUSHMAN

(B) STREET: 1615 L Street, N.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: USA

(F) ZIP: 20036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Scott, Watson T.

(B) REGISTRATION NUMBER: 26,581

(C) REFERENCE/DOCKET NUMBER: WTS/5683/84697/SRL

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 861-3000

(B) TELEFAX: (202) 822-0944

(C) TELEX: 248453 CUSH

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2229 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 126..2075

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: GGGCAATATA TCTGGAGGCC TATGAAGAAT ACACCAGCAA GCTAGATGCA CTCCAACAAA 60

GAGAACAACA GTTATTGGAA TCTCTGGGGA ACGGAACTGA TTTTTCTGTT TCTAGCTCTG 120

CATCA ATG GAT ACC GTT ACA TCT TCT TCC TCT TCT AGC CTT TCA GTG 167 Met Asp Thr Val Thr Ser Ser Ser Ser Ser Ser Leu Ser Val 1 5 10

CTA CCT TCA TCT CTT TCA GTT TTT CAA AAT CCC ACA GAT GTG GCA CGG 215 Leu Pro Ser Ser Leu Ser Val Phe Gin Asn Pro Thr Asp Val Ala Arg 15 20 25 30

AGC AAC CCC AAG TCA CCA CAA AAA CCT ATC GTT AGA GTC TTC CTG CCC 263 Ser Asn Pro Lys Ser Pro Gin Lys Pro lie Val Arg Val Phe Leu Pro 35 40 45

AAC AAA CAG AGG ACA GTG GTA CCT GCA AGG TGT GGA GTT ACA GTC CGA 311 Asn Lys Gin Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr. Val Arg 50 55 60

GAC AGT CTA AAG AAA GCA CTG ATG ATG AGA GGT CTA ATC CCA GAG TGC 359 Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu He Pro Glu Cys 65 70 75

TGT GCT GTT TAC AGA ATT CAG GAT GGA GAG AAG AAA CCA ATT GGT TGG 407 Cys Ala Val Tyr Arg He Gin Asp Gly Glu Lys Lys Pro He Gly Trp 80 85 90

GAC ACT GAT ATT TCC TGG CTT ACT GGA GAA GAA TTG CAT GTG GAA GTG 455 Asp Thr Asp He Ser Trp Leu Thr Gly Glu Glu Leu His Val Glu Val 95 100 105 110

TTG GAG AAT GTT CCA CTT ACA ACA CAC AAC TTT GTA CGA AAA ACG TTT 503 Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys Thr Phe 115 120 125

^' TTC ACC TTA GCA TTT TGT GAC TTT TGT CGA AAG CTG CTT TTC CAG GGT 551 Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe Gin Gly 130 135 140

TTC CGC TGT CAA ACA TGT GGT TAT AAA TTT CAC CAG CGT TGT AGT ACA 599 Phe Arg Cys Gin Thr Cys Gly Tyr Lys Phe His Gin Arg Cys Ser Thr 145 150 155

GAA GTT CCA CTG ATG TGT GTT AAT TAT GAC CAA CTT GAT TTG CTG TTT 647 Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gin Leu Asp Leu Leu Phe 160 165 170

GTC TCC AAG TTC TTT GAA CAC CAC CCA ATA CCA CAG GAA GAG GCG TCC 695 Val Ser Lys Phe Phe Glu His His Pro He Pro Gin Glu Glu Ala Ser 175 180 185 190

TTA GCA GAG ACT GCC CTA ACA TCT GGA TCA TCC CCT TCC GCA CCC GCC 743 Leu Ala Glu Thr Ala Leu Thr Ser Gly Ser Ser Pro Ser Ala Pro Ala 195 200 205

TCG GAC TCT ATT GGG CCC CAA ATT CTC ACC AGT CCG TCT CCT TCA AAA 791 Ser Asp Ser He Gly Pro Gin He Leu Thr Ser Pro Ser Pro Ser Lys 210 215 220

TCC ATT CCA ATT CCA CAG CCC TTC CGA CCA GCA GAT GAA GAT CAT CGA 839 Ser He Pro He Pro Gin Pro Phe Arg Pro Ala Asp Glu Asp His Arg 225 230 235

AAT CAA TTT GGG CAA CGA GAC CGA TCC TCA TCA GCT CCC AAT GTG CAT 887 Asn Gin Phe Gly Gin Arg Asp Arg Ser Ser Ser Ala Pro Asn Val His 240 245 250

ATA AAC ACA ATA GAA CCT GTC AAT ATT GAT GAC TTG ATT AGA GAC CAA 935 He Asn Thr He Glu Pro Val Asn He Asp Asp Leu He Arg Asp Gin 255 260 265 270

GGA TTT CGT GGT GAT GGA GGA TCA ACC ACA GGT TTG TCT GCT ACC CCC 983 Gly Phe Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala Thr Pro 275 280 285

CCT GCC TCA TTA CCT GGC TCA CTA ACT AAC GTG AAA GCC TTA CAG AAA 1031 Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu Gin Lys 290 295 300

TCT CCA GGA CCT CAG CGA GAA AGG AAG TCA TCT TCA TCC TCA GAA GAC 1079 Ser Pro Gly Pro Gin Arg Glu Arg Lys Ser Ser Ser Ser Ser Glu Asp 305 310 315

AGG AAT CGA ATG AAA ACA CTT GGT AGA CGG GAC TCG AGT GAT GAT TGG 1127 Arg Asn Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp 320 325 330

GAG ATT CCT GAT GGG CAG ATT ACA GTG GGA CAA AGA ATT GGA TCT GGA 1175 Glu He Pro Asp Gly Gin He Thr Val Gly Gin Arg He Gly Ser Gly

335 340 345 350

TCA TTT GGA ACA GTC TAC AAG GGA AAG TGG CAT GGT GAT GTG GCA GTG 1223 Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val 355 360 365

AAA ATG TTG AAT GTG ACA GCA CCT ACA CCT CAG CAG TTA CAA GCC TTC 1271 Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gin Gin Leu Gin Ala Phe 370 375 380

AAA AAT GAA GTA GGA GTA CTC AGG AAA ACA CGA CAT GTG AAT ATC CTA 1319 Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn He Leu 385 390 395

CTC TTC ATG GGC TAT TCC ACA AAG CCA CAA CTG GCT ATT GTT ACC CAG 1367 Leu Phe Met Gly Tyr Ser Thr Lys Pro Gin Leu Ala He Val Thr Gin 400 405 410

TGG TGT GAG GGC TCC AGC TTG TAT CAC CAT CTC CAT ATC ATT GAG ACC 1415 Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His He He Glu Thr 415 420 425 430

AAA TTT GAG ATG ATC AAA CTT ATA GAT ATT GCA CGA CAG ACT GCA CAG 1463 Lys Phe Glu Met He Lys Leu He Asp He Ala Arg Gin Thr Ala Gin 435 440 445 GGC ATG GAT TAC TTA CAC GCC AAG TCA ATC ATC CAC AGA GAC CTC AAG 1511 Gly Met Asp Tyr Leu His Ala Lys Ser He He His Arg Asp Leu Lys 450 455 460

AGT AAT AAT ATA TTT CTT CAT GAA GAC CTC ACA GTA AAA ATA GGT GAT 1559 Ser Asn Asn He Phe Leu His Glu Asp Leu Thr Val Lys He Gly Asp 465 470 475

TTT GGT CTA GCT ACA GTG AAA TCT CGA TGG AGT GGG TCC CAT CAG TTT 1607 Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gin Phe 480 485 490

GAA CAG TTG TCT GGA TCC ATT TTG TGG ATG GCA CCA GAA GTC ATC AGA 1655 Glu Gin Leu Ser Gly Ser He Leu Trp Met Ala Pro Glu Val He Arg 495 500 505 510

ATG CAA GAT AAA AAT CCA TAC AGC TTT CAG TCA GAT GTA TAT GCA TTT 1703 Met Gin Asp Lys Asn Pro Tyr Ser Phe Gin Ser Asp Val Tyr Ala Phe 515 520 525

GGG ATT GTT CTG TAT GAA TTG ATG ACT GGA CAG TTA CCT TAT TCA AAC 1751 Gly He Val Leu Tyr Glu Leu Met Thr Gly Gin Leu Pro Tyr Ser Asn 530 535 540

ATC AAC AAC AGG GAC CAG ATA ATT TTT ATG GTG GGA CGA GGA TAC CTG 1799 He Asn Asn Arg Asp Gin He He Phe Met Val Gly Arg Gly Tyr Leu 545 550 555

TCT CCA GAT CTC AGT AAG GTA CGG AGT AAC TGT CCA AAA GCC ATG AAG 1847 Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala Met Lys 560 565 570

AGA TTA ATG GCA GAG TGC CTC AAA AAG AAA AGA GAT GAG AGA CCA CTC 1895 Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg Pro Leu 575 580 585 590

TTT CCC CAA ATT CTC GCC TCT ATT GAG CTG CTG GCC CGC TCA TTG CCA 1943 Phe Pro Gin He Leu Ala Ser He Glu Leu Leu Ala Arg Ser Leu Pro 595 600 605

AAA ATT CAC CGC AGT GCA TCA GAA CCC TCC TTG AAT CGG GCT GGT TTC 1991 Lys He His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala Gly Phe 610 615 620

CAA ACA GAG GAT TTT AGT CTA TAT GCT TGT GCT TCT CCA AAA ACA CCC 2039 Gin Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys Thr Pro 625 630 635

ATC CAG GCA GGG GGA TAT GGT GCG TTT CCT GTC CAC TGAAACAAAT 2085 He Gin Ala Gly Gly Tyr Gly Ala Phe Pro Val His 640 645 650

GAGTGAGAGA GTTCAGGAGA GTAGCAACAA AAGGAAAATA AATGAACATA TGTTTGCTTA 2145

TATGTTAAAT TGAATAAAAT ACTCTCTTTT TTTTTAAGGT GGAAAAAAAA AAAAAAAAAA 2205

AAAAAAAAAA AAAAAAAAAA ACCC 2229 (2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 650 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Met Asp Thr Val Thr Ser Ser Ser Ser Ser Ser Leu Ser Val Leu Pro 1 5 10 15

Ser Ser Leu Ser Val Phe Gin Asn Pro Thr Asp Val Ala Arg Ser Asn 20 25 30

Pro Lys Ser Pro Gin Lys Pro He Val Arg Val Phe Leu Pro Asn Lys 35 40 45

Gin Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr Val Arg Asp Ser 50 55 60

Leu Lys Lys Ala Leu Met Met Arg Gly Leu He Pro Glu Cys Cys Ala 65 70 75 80

Val Tyr Arg He Gin Asp Gly Glu Lys Lys Pro He Gly Trp Asp Thr 85 90 95

Asp He Ser Trp Leu Thr Gly Glu Glu Leu His Val Glu Val Leu Glu 100 105 110

Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys Thr Phe Phe Thr 115 120 125

Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe Gin Gly Phe Arg 130 135 140

Cys Gin Thr Cys Gly Tyr Lys Phe His Gin Arg Cys Ser Thr Glu Val 145 150 155 160

Pro Leu Met Cys Val Asn Tyr Asp Gin Leu Asp Leu Leu Phe Val Ser 165 170 175

Lys Phe Phe Glu His His Pro He Pro Gin Glu Glu Ala Ser Leu Ala 180 185 190

Glu Thr Ala Leu Thr Ser Gly Ser Ser Pro Ser Ala Pro Ala Ser Asp 195 200 205

Ser He Gly Pro Gin He Leu Thr Ser Pro Ser Pro Ser Lys Ser He 210 215 220

Pro He Pro Gin Pro Phe Arg Pro Ala Asp Glu Asp His Arg Asn Gin 225 230 235 240 Phe Gly Gin Arg Asp Arg Ser Ser Ser Ala Pro Asn Val His He Asn 245 250 255

Thr He Glu Pro Val Asn He Asp Asp Leu He Arg Asp Gin Gly Phe 260 265 270

Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala Thr Pro Pro Ala 275 280 285

Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu Gin Lys Ser Pro 290 295 300

Gly Pro Gin Arg Glu Arg Lys Ser Ser Ser Ser Ser Glu Asp Arg Asn 305 310 315 320

Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu He 325 330 335

Pro Asp Gly Gin He Thr Val Gly Gin Arg He Gly Ser Gly Ser Phe 340 345 350

Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val Lys Met 355 360 365

Leu Asn Val Thr Ala Pro Thr Pro Gin Gin Leu Gin Ala Phe Lys Asn 370 375 380

Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn He Leu Leu Phe 385 390 395 400

Met Gly Tyr Ser Thr Lys Pro Gin Leu Ala He Val Thr Gin Trp Cys 405 410 415

Glu Gly Ser Ser Leu Tyr His His Leu His He He Glu Thr Lys Phe 420 425 430

Glu Met He Lys Leu He Asp He Ala Arg Gin Thr Ala Gin Gly Met 435 440 445

Asp Tyr Leu His Ala Lys Ser He He His Arg Asp Leu Lys Ser Asn 450 455 460

Asn He Phe Leu His Glu Asp Leu Thr Val Lys He Gly Asp Phe Gly 465 470 475 480

Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gin Phe Glu Gin 485 490 495

Leu Ser Gly Ser He Leu Trp Met Ala Pro Glu Val He Arg Met Gin 500 505 510 sp Lys Asn Pro Tyr Ser Phe Gin Ser Asp Val Tyr Ala Phe Gly He 515 520 525 Val Leu Tyr Glu Leu Met Thr Gly Gin Leu Pro Tyr Ser Asn He Asn 530 535 540

Asn Arg Asp Gin He He Phe Met Val Gly Arg Gly Tyr Leu Ser Pro 545 550 555 560

Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala Met Lys Arg Leu 565 570 575

Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg Pro Leu Phe Pro 580 585 590

Gin He Leu Ala Ser He Glu Leu Leu Ala Arg Ser Leu Pro Lys He 595 600 605

His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala Gly Phe Gin Thr 610 615 620

Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys Thr Pro He Gin 625 630 635 640

Ala Gly Gly Tyr Gly Ala Phe Pro Val His 645 650

Claims

WHAT IS CLAIMED IS:

1. A DNA segment coding for a polypeptide having an amino acid sequence corresponding to a B-raf protein kinase.

2. The DNA segment according to claim 1 , wherein said DNA segment has the sequence shown in SEQ ID N0:1 or allelic or species variation thereof.

3. The DNA segment according to claim 1 , wherein said DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2 or allelic or species variation thereof.

4. A polypeptide free of proteins with which it is naturally associated.and having an amino acid sequence corresponding to a B-raf protein kinase.

5. A polypeptide bound to a solid support and having an amino acid sequence corresponding to a B-raf protein kinase.

6. The polypeptide according to claim 4, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:2 or allelic or species variation thereof.

7. The polypeptide according to claim 5, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:2 or allelic or species variation thereof.

8. A recombinant DNA molecule comprising a vector and the DNA segment according to claim 1.

9. A cell that contains the recombinant DNA molecule according to claim 8.

10. A method of producing a polypeptide having an amino acid sequence corresponding to a B-raf protein kinase comprising culturing the cell according to claim 9 under conditions such that said DNA segment is expressed and said polypeptide thereby produced, and isolating said polypeptide.

11. A hybridoma which produces a B-raf protein kinase specific monoclonal antibody.

12. The hybridoma according to claim 11, wherein said hybridoma results from the fusion of a myeloma cell and a spleen cell.

13. The hybridoma according to claim 12 wherein said myeloma cell is derived from a mouse.

14. The hybridoma according to claim 12 wherein said spleen cell is derived from a mammal immunized with a recombinant B-raf protein.

15. The hybridoma according to claim 14 wherein said mammal is a mouse.

16. A hybridoma producing monoclonal antibody B-raf 12S.

17. A B-raf protein kinase specific monoclonal antibody, or binding fragment thereof.

18. A monoclonal antibody having the binding characteristics of the monoclonal antibody produced by the hybridoma according to claim 16, or binding fragment thereof.

19. A diagnostic kit comprising: i) at least one monoclonal antibody according to claim 17, and ii) a conjugate comprising a binding partner of said monoclonal antibody and a label.

20. A diagnostic kit comprising a conjugate comprising: i) at least one monoclonal antibody according to claim 17, and ii) a label.

21. A diagnostic kit comprising: i) at least one monoclonal antibody according to claim 18 and ii) a conjugate comprising a binding partner of said monoclonal antibody and a label.

22. A diagnostic kit comprising a conjugate comprising: i) at least one monoclonal antibody according to claim 18 and ii) a label.