WO1997025345A1 - Fusion proteins comprising cell cycle regulatory proteins - Google Patents
Fusion proteins comprising cell cycle regulatory proteins Download PDFInfo
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- WO1997025345A1 WO1997025345A1 PCT/US1997/000140 US9700140W WO9725345A1 WO 1997025345 A1 WO1997025345 A1 WO 1997025345A1 US 9700140 W US9700140 W US 9700140W WO 9725345 A1 WO9725345 A1 WO 9725345A1
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- fusion proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
Abstract
The present invention provides novel fusion proteins comprising cyclins and CDKs. A preferred embodiment of the invention provides fusion proteins comprising human cyclin D1 and human CDK4. The fusion proteins of the invention optionally contain modifications, which facilitate their purification. Addition of histidine residues to selected constructs allows purification via immobilized metal affinity chromatography. Antigenic determinants allowing monoclonal antibody-based affinity chromatography purification are provided in selected embodiments of the invention. Protease cleavage sites are incorporated in selected constructs to allow cleavage of the regions incorporated in the cyclin-CDK fusion proteins for purification. Additional modifications which facilitate purification include strepavadin binding domains and antigenic determinants for antibody affinity chromatography.
Description
Title
FUSION PROTEINS COMPRISING CELL CYCLE REGULATORY PROTEINS
Background of the Invention
The pivotal roles which cyclins and cyclin dependent kinases play in cell cycle regulation is well established. The initial interest in cyclins resulted from observations that this family of molecules accumulated and then disappeared at precise points in the cell cycles of embryonic cells. Evans, T. et al . , Cell 33, 389-396. (1983) . Cyclin-dependent protein kinase (CDK) activation requires cyclin binding and phosphorylation of a threonine residue by the CDK-activating kinase, CAK. Several recent review articles (Norbury, c. and Nurse, P. A. Rev. Biochem. 61, 441- 470 (1992) ; Nasmyth, K. Curr. Opin. Cell Biol. 5, 166-179 (1993) and Sherr, C. J. Cell 73, 1059-1065 (1993)) detail the regulatory roles which the cyclins and the cyclin dependent kinases play in cell cycle progression. The criticality of proper cell cycle regulation is intuitive. Disruption of cell cycle regulation leads to uncontrolled cell division. Appreciation of the important roles which cyclins and cyclin dependent kinases play in cell cycle regulation has focused intense research efforts aimed at better understanding cell cycle regulation and then exploiting this knowledge for discovery and development of oncoltyics .
Exploitation of the current knowledge regarding cyclins and CDKs requires experiments involving the addition of appropriate amounts of cyclins and CDKs to allow formation of the desired cyclin-CDK complex for phosphorylation of the conserved threonine residue of the CDK prior to attempting to modulate CDK-mediated phosphorylation of the retinoblastoma protein, Rb. The stochiometric problems inherent in such complicated experimental designs are substantial. The present invention addresses this problem by providing fusion proteins comprising cyclins and CDK4. The biological activities of
these fusion proteins eliminates the stochiometry related problems.
Summary of the Invention The present invention provides novel fusion proteins comprising cyclins and CDKs. A preferred embodiement of the invention provides fusion proteins comprising human cyclin Dl and human CDK4. The fusion proteins of the invention optionally contain modifications, which facilitate their purification. Addition of histidine residues to selected constructs allows purification via immobilized metal affinity chromatography. Antigenic determinants allowing monoclonal antibody-based affinity chromatography purification are provided in selected embodiments of the invention. Proetease cleavage sites are incorporated in selected constructs to allow cleavage of the regions incorporated in the cyclin-CDK fusion proteins for purification. Additional modifications which fctcilitate purification include strepavadin binding domains and antigenic determinants for antibody affinity chromatography.
Brief Description of the Figures Figure 1 is a restriction site and function map of plasmid PK415. Figure 2 is a restriction site and function map of plasmid PK485.
Figure 3 is a restriction site and function map of plasmid pK480.
Detailed Description of the Invention
The fusion proteins of the present invention comprise cyclins and CDKs linked via various peptide spacers and optionally contain amino acid sequences, which are incorporated to facilitate purification. The DNA sequence (SEQ ID NO:l) encoding a preferred embodiment of the present invention is provided below. 1 GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA
51 ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG
101 AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA
151 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT
201 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT
251 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG
301 CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC
351 AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT
401 GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG
451 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG
501 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT
551 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA
601 ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG
651 CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT
701 GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA
751 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT
801 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC
851 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG
901 GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT
951 GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC
1001 TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA
1051 AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT
1101 TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA
1151 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT
1201 CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG
1251 CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT
1301 TGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG ■3TAACTGGCT
1351 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA
1401 GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT
1451 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG
1501 GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA
1551 ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA
1601 ACTGAGATAC CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG
1651 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG
1701 CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT
1751 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG
1801 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC
1851 CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC
1901 TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT GATACCGCTC
1951 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAA
2001 GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA
2051 ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA
2101 ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC
2151 TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT
2201 TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTT ACGGCGCGCC
2251 GCCGCCACCA TGGCGGAGGA GCAGAAGCTG ATATCCGAGG AGGACCTGCT
2301 GCTAGCAATG GAACACCAGC TCCTGTGCTG CGAAGTGGAA ACCATCCGCC
2351 GCGCGTACCC CGATGCCAAC CTCCTCAACG ACCGGGTGCT GCGGGCCATG
2401 CTGAAGGCGG AGGAGACCTG CGCGCCCTCG GTGTCCTACT TCAAATGTGT
2451 GCAAAAGGAG GTCCTGCCGT CCATGCGGAA GATCGTCGCC ACCTGGATGC
2501 TGGAGGTCTG CGAGGAACAG AAGTGCGAGG AGGAGGTCTT CCCGCTGGCC
2551 ATGAACTACC TGGACCGCTT CCTGTCGCTG GAGCCCGTGA AAAAGAGCCG
2601 CCTGCAGCTG CTGGGGGCCA CTTGCATGTT CGTGGCCTCT AAGATGAAGG
2651 AGACCATCCC CCTGACGGCC GAGAAGCTGT GCATCTACAC CGACAACTCC
2701 ATCCGGCCCG AGGAGCTGCT GCAAATGGAG CTGCTCCTGG TGAACAAGCT
2751 CAAGTGGAAC CTGGCCGCAA TGACCCCGCA CGATTTCATT GAACACTTCC
2801 TCTCCAAAAT GCCAGAGGCG GAGGAGAACA AACAGATCAT CCGCAAACAC
2851 GCGCAGACCT TCGTTGCCCT CTGTGCCACA GATGTGAAGT TCATTTCCAA
2901 TCCGCCCTCC ATGGTGGCAG CGGGGAGCGT GGTGGCCGCA GTGCAAGGCC
2951 TGAACCTGAG GAGCCCCAAC AACTTCCTGT CCTACTACCG CCTCACACGC
3001 TTCCTCTCCA GAGTGATCAA GTGTGACCCA GACTGCCTCC GGGCCTGCCA
3051 GGAGCAGATC GAAGCCCTGC TGGAGTCAAG CCTGCGCCAG GCCCAGCAGA
3101 ACATGGACCC CAAGGCCGCC GAGGAGGAGG AGGAGGAAGA GGAGGAAGAG
3151 GAGGTGGACC TGGCTTGCAC ACCCACCGAC GTGCGGGACG TGGACATCGC
3201 ATCGAAGGGT GGTGGAGGTT CTGGAGGTGG AGGATCCGGT GGTGGAGGTT
3251 CGATGGCTAC CTCTCGATAT GAGCCAGTGG CTGAAATTGG TGTCGGTGCC
3301 TATGGGACAG TGTACAAGGC CCGTGATCCC CACAGTGGCC ACTTTGTGGC
3351 CCTCAAGAGT GTGAGAGTCC CCAATGGAGG AGGAGGTGGA GGAGGCCTTC
3401 CCATCAGCAC AGTTCGTGAG GTGGCTTTAC TGAGGCGACT GGAGGCTTTT
3451 GAGCATCCCA ATGTTGTCCG GCTGATGGAC GTCTGTGCCA CATCCCGAAC
3501 TGACCGGGAG ATCAAGGTAA CCCTGGTGTT TGAGCATGTA GACCAGGACC
3551 TAAGGACATA TCTGGACAAG GCACCCCCAC CAGGCTTGCC AGCCGAAACG
3601 ATCAAGGATC TGATGCGCCA GTTTCTAAGA GGCCTAGATT TCCTTCATGC
3651 CAATTGCATC GTTCACCGAG ATCTGAAGCC AGAGAACATT CTGGTGACAA
3701 GTGGTGGAAC AGTCAAGCTG GCTGACTTTG GCCTGGCCAG AATCTACAGC
3751 TACCAGATGG CACTTACACC CGTGGTTGTT ACACTCTGGT ACCGAGCTCC
3801 CGAAGTTCTT CTGCAGTCCA CATATGCAAC ACCTGTGGAC ATGTGGAGTG
3851 TTGGCTGTAT CTTTGCAGAG ATGTTTCGTC GAAAGCCTCT CTTCTGTGGA
3901 AACTCTGAAG CCGACCAGTT GGGCAAAATC TTTGACCTGA TTGGGCTGCC
3951 TCCAGAGGAT GACTGGCCTC GAGATGTATC CCTGCCCCGT GGAGCCTTTC
4001 CCCCCAGAGG GCCCCGCCCA GTGCAGTCGG TGGTACCTGA GATGGAGGAG
4051 TCGGGAGCAC AGCTGCTGCT GGAAATGCTG ACTTTTAACC CACACAAGCG
4101 AATCTCTGCC TTTCGAGCTC TGCAGCACTC TTATCTACAT AAGGATGAAG
4151 GTAATCCGGA GGGCGGCAGC GCTTGGCGCC ACCCACAGTT CGGTGGTTGA
4201 ATAAATAGAT GAATGACCTG CAGGTTCACT GGCCGTCGTT TTACAACGTC
4251 GTGACTGGGA AAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT
4301 CCCCCTTTCG CCAGCTGGCG TAATAGCGAA GAGGCCCGCA CCGATCGCCC
4351 TTCCCAACAG TTGCGCAGCC TGAATGGCGA ATGGCGCCTG ATGCGGTATT
4401 TTCTCCTTAC GCATCTGTGC GGTATTTCAC ACCGCATATG GTGCACTCTC
4451 AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC
4501 AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG GCATCCGCTT
4551 ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA GAGGTTTTCA
4601 CCGTCATCAC CGAAACGCGC GA
The polypeptide encoded by SEQ ID N0:1 is presented below as SEQ ID NO:2.
1 MTMITPSLRR AAATMAEEQK LISEEDLLLA MEHQLLCCEV ETIRRAYPDA
51 NLLNDRVLRA MLKAEETCAP SVSYFKCVQK EVLPSMRKIV ΛT MLEVCEE
101 QKCEEEVFPL AMNYLDRFLS LEPVKKSRLQ LLGATCMFVA SKMKETIPLT
151 AEKLCIYTDN SIRPEELLQM ELLLVNKLKW NLAAMTPHDF EHFLSKMPE
201 AEENKQIIRK HAQTFVALCA TDVKFISNPP SMVAAGSWA AVQGLNLRSP
251 NNFLSYYRLT RFLSRVIKCD PDCLRACQEQ IEALLESSLR QAQQNMDPKA
301 AEEEEEEEEE EEVDLACTPT DVRDVDIASK GGGGSGGGGS GGGGSMATSR
351 YEPVAEIGVG AYGTVYKARD PHSGHFVALK SVRVPNGGGG GGGLPISTVR
401 EVALLRRLEA FEHPNWRLM DVCATSRTDR EIKVTLVFEH VDQDLRTYLD
451 KAPPPGLPAE TIKDLMRQFL RGLDFLHANC IVHRDLKPEN LVTSGGTVK
501 LADFGLARIY SYQMALTPW VTL YRAPEV LLQSTYATPV DM SVGCIFA
551 EMFRRKPLFC GNSEADQLGK IFDLIGLPPE DDWPRDVSLP RGAFPPRGPR
601 PVQSWPEME ESGAQLLLEM LTFNPHKRIS AFRALQHSYL HKDEGNPEGG
651 SAWRHPQFGG
The DNA sequence of SEQ ID NO:l is the preferred coding sequence for the polypeptide of SEQ ID NO:2. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:2 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:2 are
contemplated by the present invention and thus are within the scope of the present invention.
The DNA sequence of SEQ ID N0:1 is a component of the plasmid K415. A restriction site and function map of plasmid K415 is provided in Figure 1. E. coli host cells transformed with K415 were deposited in the NRRL, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country. The NRRL accession number for E. coli/K415 is B-21490. The routine nature of culturing such organisms, preparing plasmids from the transformants, digesting the plasmids with appropriate restriction endonucleases and isolating the appropriate DNA fragment obviate the need or desirability of discussing these routine steps.
The distinct functional subcomponents of the polypeptide of SEQ ID NO:2 are described by reference to the amino acid residue numbers provided in SEQ ID NO:2. Residues 18 through 27 comprise the epitope recognized by the monoclonal antibody designated myc. Residues 31 though 327 correspond to human cyclin Dl . Residues 331 through 345 are an illustrative "linker" or polypedtide connector. The terms "linker", "polypeptide connector" and "hinge" are used interchangeably in describing the present invention and all three terms refer to the sequences of amino acids which are used to connect the cyclin and CDK components of the fusion proteins of the present invention. Residues 346 through 648 correspond to human CDK4. Residues 651 through 660 correspond to strepavadin and were engineered into the molecule to allow facile purification.
The polypeptide of SEQ ID NO:2 has numerous components which allow great flexibility in purification, but are not required for the ultimate benefit provided by the present invention-a biologically active fusion protein comprising cyclin and CDK components . A most preferred aspect of this embodiment of the present invention is the
cyclin Dl-linker-CDK4 component of the molecule. This most preferred aspect is provided below as SEQ ID NO:3.
41 MEHQLLCCEV ETIRRAYPDA
51 NLLNDRVLRA MLKAEETCAP SVSYFKCVQK EVLPSMRKIV ATWMLEVCEE
101 QKCEEEVFPL AMNYLDRFLS LEPVKKSRLQ LLGATCMFVA SKMKETIPLT
151 AEKLCIYTDN SIRPEELLQM ELLLVNKLKW NLAAMTPHDF IEHFLSKMPE
201 AEENKQIIRK HAQTFVALCA TDVKFISNPP SMVAAGSWA AVQGLNLRSP
251 NNFLSYYRLT RFLSRVIKCD PDCLRACQEQ lEALLESSLR QAQQNMDPKA
301 AEEEEEEEEE EEVDLACTPT DVRDVDIASK GGGGSGGGGS GGGGSMATSR
351 YEPVAEIGVG AYGTVYKARD PHSGHFVALK SVRVPNGGGG GGGLPISTVR
401 EVALLRRLEA FEHPNWRLM DVCATSRTDR EIKVTLVFEH VDQDLRTYLD
451 KAPPPGLPAE TIKDLMRQFL RGLDFLHANC IVHRDLKPEN ILVTSGGTVK
501 LADFGLARIY SYQMALTPW VTLWYRAPEV LLQSTYATPV DM SVGCIFA
551 EMFRRKPLFC GNSEADQLGK IFDLIGLPPE DDWPRDVSLP RGAFPPRGPR
601 PVQSWPEME ESGAQLLLEM LTFNPHKRIS AFRALQHSYL HKDEGNPE
Biologically active fusion protein comprising a member of the cyclin family and the CDK family are further illustrated by the DNA sequence of SEQ ID NO: and the corresponding polypeptide sequence, SEQ ID NO:5. SEQ ID NO:4 is provided immediately below.
1 GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA
51 ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG
101 AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA
151 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT
201 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT
251 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG
301 CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC
351 AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT
401 GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG
451 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG
501 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT
551 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA
601 ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG
651 CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT
701 GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA
751 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT
801 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC
851 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG
901 GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT
951 GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC
1001 TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA
1051 AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT
1101 TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA
1151 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT
1201 CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG
1251 CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT
1301 TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT
1351 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA
1401 GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT
1451 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG
1501 GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA
1551 ACGGGGGGTT CGTGCACAC GCCCAGCTTG GAGCGAACGA CCTACACCGA
1601 ACTGAGATAC CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG
1651 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG
1701 CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT
1751 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG
1801 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC
1851 CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC
1901 TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT GATACCGCTC
1951 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAA
2001 GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA
2051 ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA
2101 ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC
2151 TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT
2201 TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTT ACGGCGCGCC
2251 GCCGCCACCA TGGCGCATCA TCATCATCAT CATGGAGGTG GAGGTTCGGA
2301 GCAGAAGCTT ATTTCCGAGG AGGATCTGCT GGTGCCACGC GGTTCCCTGC
2351 TAGCAATGGA ACACCAGCTC CTGTGCTGCG AAGTGGAAAC CATCCGCCGC
2401 GCGTACCCCG ATGCCAACCT CCTCAACGAC CGGGTGCTGC GGGCCATGCT
2451 AAAGGCGGAG GAGACCTGCG CGCCCTCGGT GTCCTACTTC AAATGTGTGC
2501 AAAAGGAGGT CCTGCCGTCC ATGCGGAAGA TCGTCGCCAC CTGGATGCTG
2551 GAGGTCTGCG AGGAACAGAA GTGCGAGGAG GAGGTCTTCC CGCTGGCCAT
2601 GAACTACCTG GACCGCTTCC TGTCGCTGGA GCCCGTGAAA AAGAGCCGCC
2651 TGCAGCTGCT GGGGGCCACT TGCATGTTCG TGGCCTCTAA GATGAAGGAG
2701 ACCATCCCCC TGACGGCCGA GAAGCTGTGC ATCTACACCG ACAACTCCAT
2751 CCGGCCCGAG GAGCTGCTGC AAATGGAGCT GCTCCTGGTG AACAAGCTCA
2801 AGTGGAACCT GGCCGCAATG ACCCCGCACG ATTTCATTGA ACACTTCCTC
2851 TCCAAAATGC CAGAGGCGGA GGAGAACAAA CAGATCATCC GCAAACACGC
2901 GCAGACCTTC GTTGCCCTCT GTGCCACAGA TGTGAAGTTC ATTTCCAATC
2951 CGCCCTCCAT GGTGGCAGCG GGGAGCGTGG TGGCCGCAGT GCAAGGCCTG
3001 AACCTGAGGA GCCCCAACAA CTTCCTGTCC TACTACCGCC TCACACGCTT
3051 CCTCTCCAGA GTGATCAAGT GTGACCCAGA CTGCCTCCGG GCCTGCCAGG
3101 AGCAGATCGA AGCCCTGCTG GAGTCAAGCC TGCGCCAGGC CCAGCAGAAC
3151 ATGGACCCCA AGGCCGCCGA GGAGGAGGAG GAGGAAGAGG AGGAAGAGGA
3201 GGTGGACCTG GCTTGCACAC CCACCGACGT GCGGGACGTG GACATCGCAT
3251 CGAAGGGTGG TGGAGGTTCT GGAGGTGGAG GATCCGGTGG TGGAGGTTCG
3301 ATGGCTACCT CTCGATATGA GCCAGTGGCT GAAATTGGTG TCGGTGCCTA
3351 TGGGACAGTG TACAAGGCCC GTGATCCCCA CAGTGGCCAC TTTGTGGCCC
3401 TCAAGAGTGT GAGAGTCCCC AATGGAGGAG GAGGTGGAGG AGGCCTTCCC
3451 ATCAGCACAG TTCGTGAGGT GGCTTTACTG AGGCGACTGG AGGCTTTTGA
3501 GCATCCCAAT GTTGTCCGGC TGATGGACGT CTGTGCCACA TCCCGAACTG
3551 ACCGGGAGAT CAAGGTAACC CTGGTGTTTG AGCATGTAGA CCAGGACCTA
3601 AGGACATATC TGGACAAGGC ACCCCCACCA GGCTTGCCAG CCGAAACGAT
3651 CAAGGATCTG ATGCGCCAGT TTCTAAGAGG CCTAGATTTC CTTCATGCCA
3701 ATTGCATCGT TCACCGAGAT CTGAAGCCAG AGAACATTCT GGTGACAAGT
3751 GGTGGAACAG TCAAGCTGGC TGACTTTGGC CTGGCCAGAA TCTACAGCTA
3801 CCAGATGGCA CTTACACCCG TGGTTGTTAC ACTCTGGTAC CGAGCTCCCG
3851 AAGTTCTTCT GCAGTCCACA TATGCAACAC CTGTGGACAT GTGGAGTGTT
3901 GGCTGTATCT TTGCAGAGAT GTTTCGTCGA AAGCCTCTCT TCTGTGGAAA
3951 CTCTGAAGCC GACCAGTTGG GCAAAATCTT TGACCTGATT GGGCTGCCTC
4001 CAGAGGATGA CTGGCCTCGA GATGTATCCC TGCCCCGTGG AGCCTTTCCC
4051 CCCAGAGGGC CCCGCCCAGT GCAGTCGGTG GTACCTGAGA TGGAGGAGTC
4101 GGGAGCACAG CTGCTGCTGG AAATGCTGAC TTTTAACCCA CACAAGCGAA
4151 TCTCTGCCTT TCGAGCTCTG CAGCACTCTT ATCTACATAA GGATGAAGGT
4201 AATCCGGAGG GCGGCAGCGC TTGGCGCCAC CCACAGTTCG GTGGTTGAAT
4251 AAATAGATGA ATGACCTGCA GGTGCACTCT CAGTACAATC TGCTCTGATG
4301 CCGCATAGTT AAGCCAGCCC CGACACCCGC CAACACCCGC TGACGCGCCC
4351 TGACGGGCTT GTCTGCTCCC GGCATCCGCT TACAGACAAG CTGTGACCGT
4401 CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA CCGAAACGCG
4451 CGA
The polypeptide encoded by the sequence of SEQ ID NO:4 is provided below as SEQ ID NO:5.
1 MAHHHHHHGG GGSEQKLISE EDLLVPRGSL LAMEHQLLCC EVETIRRAYP
51 DANLLNDRVL RAMLKAEETC APSVSYFKCV QKEVLPSMRK IVATWMLEVC
101 EEQKCEEEVF PLAMNYLDRF LSLEPVKKSR LQLLGATCMF VASKMKETIP
151 LTAEKLCIYT DNSIRPEELL QMELLLVNKL KWNLAAMTPH DFIEHFLSKM
201 PEAEENKQII RKHAQTFVAL CATDVKFISN PPSMVAAGSV VAAVQGLNLR
251 SPNNFLSYYR LTRFLSRVIK CDPDCLRACQ EQIEALLESS LRQAQQNMDP
301 KAAEEEEEEE EEEEVDLACT PTDVRDVDIA SKGGGGSGGG GSGGGGSMAT
351 SRYEPVAEIG VGAYGTVYKA RDPHSGHFVA LKSVRVPNGG GGGGGLPIST
401 VREVALLRRL EAFEHPNWR LMDVCATSRT DREIKVTLVF EHVDQDLRTY
451 LDKAPPPGLP AETIKDLMRQ FLRGLDFLHA NCIVHRDLKP ENILVTSGGT
501 VKLADFGLAR IYSYQMALTP VWTLWYRAP EVLLQSTYAT PVDMWSVGCI
551 FAEMFRRKPL FCGNSEADQL GKIFDLIGLP PEDDWPRDVS LPRGAFPPRG
601 PRPVQSWPE MEESGAQLLL EMLTFNPHKR ISAFRALQHS YLHKDEGNPE
651 GGSAWRHPQF GG
The DNA sequence of SEQ ID NO:4 is the preferred coding sequence for the polypeptide of SEQ ID NO: 5. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:4 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:5 are contemplated by the present invention and thus are within the scope of the present invention.
The DNA sequence of SEQ ID NO:4 is a component of the plasmid K485. A restriction site and function map of plasmid K485 is provided in Figure 2. E. coli host cells transformed with K485 were deposited in the NRRL, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will
be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country. The NRRL accession number for E. coli/K485 is B-21492. The routine nature of culturing such organisms, preparing plasmids from the transformants, digesting the plasmids with appropriate restriction endonucleases and isolating the appropriate DNA fragment obviate the need or desirability of discussing these routine steps.
The DNA sequence of Sequence ID 4 and the polypeptide encoded thereby comprise human cyclin Dl and human CDK4 which are joined by a polypeptide linker. The distinct functional subcomponents of the polypeptide of SEQ ID NO:5 are described by reference to the amino acid residue numbers provided in SEQ ID NO:5. Amino acid residues 2 through 8 are Histidine residues which were incorporated to allow immobilized metal affinity chromatography purification. Residues 14 through 23 contain the antigenic determinant recognized by the myc monoclonal antibody and thereby allow myc monoclonal antibody based affinity purification. Residues 24 through 28 contain a thrombin cleavage site and were engineered into the polypeptide of SEQ ID NO:5 to allow cleavage of the molecule on the amino side of the human cyclin Dl component. Residues 43 through 329 correspond to human cyclin Dl. Residues 333 through 347 are the polypeptide linker used to join the human cyclin Dl and human CDK4 components of the molecule. Residues 348 through 650 correspond to human CDK4. Residues 653 through 662 were engineered into the molecule to provide a sequence which binds to paramagnetic streptavadin beads and thus allows facile purification of the molecule.
The present invention also provides the DNA sequence of SEQ ID NO:6, which is presented below.
1 GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA
51 ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG
101 AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA
151 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT
201 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT
251 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG
301 CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC
351 AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT
401 GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG
451 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG
501 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT
551 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA
601 ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG
651 CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT
701 GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA
751 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT
801 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC
851 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG
901 GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT
951 GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC
1001 TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA
1051 AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT
1101 TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA
1151 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT
1201 CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG
1251 CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT
1301 TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT
1351 TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA
1401 GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT
1451 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG
1501 GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA
1551 ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA
1601 ACTGAGATAC CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG
1651 GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG
1701 CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT
1751 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG
1801 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC
1851 CTGGCCTTTT GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC
1901 TGATTCTGTG GATAACCGTA TTACCGCCTT TGAGTGAGCT GATACCGCTC
1951 GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA GGAAGCGGAA
2001 GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA
2051 ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA
2101 ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC
2151 TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT
2201 TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTT ACGGCGCGCC
2251 GCCGCCACCA TGGCGCATCA TCATCATCAT CATGGAGGTG GAGGTTCGGA
2301 GCAGAAGCTT ATTTCCGAGG AGGATCTGCT GGTGCCACGC GGTTCCCTGC
2351 TAGCAATGGA ACACCAGCTC CTGTGCTGCG AAGTGGAAAC CATCCGCCGC
2401 GCGTACCCCG ATGCCAACCT CCTCAACGAC CGGGTGCTGC GGGCCATGCT
2451 AAAGGCGGAG GAGACCTGCG CGCCCTCGGT GTCCTACTTC AAATGTGTGC
2501 AAAAGGAGGT CCTGCCGTCC ATGCGGAAGA TCGTCGCCAC CTGGATGCTG
2551 GAGGTCTGCG AGGAACAGAA GTGCGAGGAG GAGGTCTTCC CGCTGGCCAT
2601 GAACTACCTG GACCGCTTCC TGTCGCTGGA GCCCGTGAAA AAGAGCCGCC
2651 TGCAGCTGCT GGGGGCCACT TGCATGTTCG TGGCCTCTAA GATGAAGGAG
2701 ACCATCCCCC TGACGGCCGA GAAGCTGTGC ATCTACACCG ACAACTCCAT
2751 CCGGCCCGAG GAGCTGCTGC AAATGGAGCT GCTCCTGGTG AACAAGCTCA
2801 AGTGGAACCT GGCCGCAATG ACCCCGCACG ATTTCATTGA ACACTTCCTC
2851 TCCAAAATGC CAGAGGCGGA GGAGAACAAA CAGATCATCC GCAAACACGC
2901 GCAGACCTTC GTTGCCCTCT GTGCCACAGA TGTGAAGTTC ATTTCCAATC
2951 CGCCCTCCAT GGTGGCAGCG GGGAGCGTGG TGGCCGCAGT GCAAGGCCTG
3001 AACCTGAGGA GCCCCAACAA CTTCCTGTCC TACTACCGCC TCACACGCTT
3051 CCTCTCCAGA GTGATCAAGT GTGACCCAGA CTGCCTCCGG GCCTGCCAGG
3101 AGCAGATCGA AGCCCTGCTG GAGTCAAGCC TGCGCCAGGC CCAGCAGAAC
3151 ATGGACCCCA AGGCCGCCGA GGAGGAGGAG GAGGAAGAGG AGGAAGAGGA
3201 GGTGGACCTG GCTTGCACAC CCACCGACGT GCGGGACGTG GACATCGCAT
3251 CGATGGGTGG AGGTTCTGGT GGAGGTTCTG GTGGAGGTTC TGGTGGAGGT
3301 TCTGGTGGAG GTTCTGGTGG AGGTTCTGGC TTAAGTTCGA AGGGTGGTGG
3351 AGGTTCTGGA GGTGGAGGAT CCGGTGGTGG AGGTTCGATG GCTACCTCTC
3401 GATATGAGCC AGTGGCTGAA ATTGGTGTCG GTGCCTATGG GACAGTGTAC
3451 AAGGCCCGTG ATCCCCACAG TGGCCACTTT GTGGCCCTCA AGAGTGTGAG
3501 AGTCCCCAAT GGAGGAGGAG GTGGAGGAGG CCTTCCCATC AGCACAGTTC
3551 GTGAGGTGGC TTTACTGAGG CGACTGGAGG CTTTTGAGCA TCCCAATGTT
3601 GTCCGGCTGA TGGACGTCTG TGCCACATCC CGAACTGACC GGGAGATCAA
3651 GGTAACCCTG GTGTTTGAGC ATGTAGACCA GGACCTAAGG ACATATCTGG
3701 ACAAGGCACC CCCACCAGGC TTGCCAGCCG AAACGATCAA GGATCTGATG
3751 CGCCAGTTTC TAAGAGGCCT AGATTTCCTT CATGCCAATT GCATCGTTCA
3801 CCGAGATCTG AAGCCAGAGA ACATTCTGGT GACAAGTGGT GGAACAGTCA
3851 AGCTGGCTGA CTTTGGCCTG GCCAGAATCT ACAGCTACCA GATGGCACTT
3901 ACACCCGTGG TTGTTACACT CTGGTACCGA GCTCCCGAAG TTCTTCTGCA
3951 GTCCACATAT GCAACACCTG TGGACATGTG GAGTGTTGGC TGTATCTTTG
4001 CAGAGATGTT TCGTCGAAAG CCTCTCTTCT GTGGAAACTC TGAAGCCGAC
4051 CAGTTGGGCA AAATCTTTGA CCTGATTGGG CTGCCTCCAG AGGATGACTG
4101 GCCTCGAGAT GTATCCCTGC CCCGTGGAGC CTTTCCCCCC AGAGGGCCCC
4151 GCCCAGTGCA GTCGGTGGTA CCTGAGATGG AGGAGTCGGG AGCACAGCTG
4201 CTGCTGGAAA TGCTGACTTT TAACCCACAC AAGCGAATCT CTGCCTTTCG
4251 AGCTCTGCAG CACTCTTATC TACATAAGGA TGAAGGTAAT CCGGAGGGCG
4301 GCAGCGCTTG GCGCCACCCA CAGTTCGGTG GTTGAATAAA TAGATGAATG
4351 ACCTGCAGGT GCACTCTCAG TACAATCTGC TCTGATGCCG CATAGTTAAG
4401 CCAGCCCCGA CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC
4451 TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC CGGGAGCTGC
4501 ATGTGTCAGA GGTTTTCACC GTCATCACCG AAACGCGCGA
The polypeptide encoded by SEQ ID NO:6 is presented below as SEQ ID NO:7.
1 MTMITPSLRR AAATMAHHHH HHGGGGSEQK LISEEDLLVP RGSLLAMEHQ
51 LLCCEVETIR RAYPDANLLN DRVLRAMLKA EETCAPSVSY FKCVQKEVLP
101 SMRKIVATWM LEVCEEQKCE EEVFPLAMNY LDRFLSLEPV KKSRLQLLGA
151 TCMFVASKMK ETIPLTAEKL CIYTDNSIRP EELLQMELLL VNKLKWNLAA
201 MTPHDFIEHF LSKMPEAEEN KQIIRKHAQT FVALCATDVK FISNPPSMVA
251 AGSWAAVQG LNLRSPNNFL SYYRLTRFLS RVIKCDPDCL RACQEQIEAL
301 LESSLRQAQQ NMDPKAAEEE EEEEEEEEVD LACTPTDVRD VDIASMGGGS
351 GGGSGGGSGG GSGGGSGGGS GLSSKGGGGS GGGGSGGGGS MATSRYEPVA
401 EIGVGAYGTV YKARDPHSGH FVALKSVRVP NGGGGGGGLP ISTVREVALL
451 RRLEAFEHPN WRLMDVCAT SRTDREIKVT LVFEHVDQDL RTYLDKAPPP
501 GLPAETIKDL MRQFLRGLDF LHANCIVHRD LKPENILVTS GGTVKLADFG
551 LARIYSYQMA LTPVWTLWY RAPEVLLQST YATPVDMWSV GCIFAEMFRR
601 KPLFCGNSEA DQLGKIFDLI GLPPEDDWPR DVSLPRGAFP PRGPRPVQSV
651 VPEMEESGAQ LLLEMLTFNP HKRISAFRAL QHSYLHKDEG NPEGGSAWRH
701 PQFGG
The DNA sequence of SEQ ID NO:6 is the preferred coding sequence for the polypeptide of SEQ ID NO:7. Numerous other DNA sequences will also encode the polypeptide of SEQ ID NO:6 due to the degeneracy of the genetic code. All DNA sequences encoding the polypeptide of SEQ ID NO:7 are contemplated by the present invention and thus are within the scope of the present invention.
The DNA sequence of SEQ ID NO:6 is a component of the plasmid K480. A restriction site and function map of plasmid K480 is provided in Figure 3. E. coli host cells transformed with K480 were deposited in the NRRL, Northern
Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604 on or before August 9, 1995 and will be available pursuant to Budapest Treaty requirements upon issuance of a patent in a Budapest signatory country. The NRRL accession number for E. coJi/K480 is B-21491. The routine nature of culturing such organisms, preparing plasmids from the transformants, digesting the plasmids with appropriate restriction endonucleases and isolating the appropriate DNA fragment obviate the need or desirability of discussing these routine steps.
The DNA sequence of SEQ ID NO:6 and the polypeptide encoded thereby comprise human cyclin Dl and human CDK4 which are joined by a polypeptide linker. The distinct functional subcomponents of the polypeptide of SEQ ID NO:7 are described by reference to the amino acid residue numbers provided in
SEQ ID NO:7. Amino acid residues 17 through 22 are Histidine residues which were incorporated to allow immobilized metal affinity chromatography purification. Residues 28 through 37 contain the antigenic determinant recognized by the myc monoclonal antibody and thereby allow myc monoclonal antibody based affinity purification. Residues 38 through 43 contain a thrombin cleavage site and were engineered into the polypeptide of Sequence ID 7 to allow cleavage of the molecule on the amino side of the human cyclin Dl. component. Residues 47 through 343 correspond to human cycl:.n Dl.
Residues 347 through 390 are the polypeptide linker used to join the human cyclin Dl and human CDK4 components of the molecule. Residues 391 through 693 correspond to human CDK4. Residues 696 through 705 were engineered into the molecule to provide a sequence which binds to paramagnetic streptavadin beads and thus allows facile purification of the molecule.
The molecule of SEQ ID NO:7 shares several features with the molecules of SEQ ID Nos:2 and 5. The polypeptide linker which joins the human cyclin Dl and the human CDK4 portions of the molecule of SEQ ID NO:7 is substantially different from the polypeptide linkers of the molecules of SEQ ID Nos: 2 and 5. The structural dissimilarity of the
linkers combined with the biological activity of the fusion proteins of the invention underscores the flexibility in linker selection. Accordingly, the fusion proteins of the present invention are not limited to cyclin-CDK fusion proteins containing the linkers which are specifically exemplified.
The fusion protein of SEQ ID NO:7 has the additional features discussed above for allowing great flexibility in choice of purification schemes. The preferred aspect of this embodiment of the present invention is the segment of the molecule comprising the biologically active human cyclin Dl-linker-human CDK4 sequence. This preferred sequence is set forth below as SEQ ID NO:8.
47 MEHQ
51 LLCCEVETIR RAYPDANLLN DRVLRAMLKA EETCAPSVSY FKCVQKEVLP
101 SMRKIVATWM LEVCEEQKCE EEVFPLAMNY LDRFLSLEPV KKSRLQLLGA
151 TCMFVASKMK ETIPLTAEKL CIYTDNSIRP EELLQMELLL VNKLKWNLAA
201 MTPHDFIEHF LSKMPEAEEN KQIIRKHAQT FVALCATDVK FISNPPSMVA
251 AGSWAAVQG LNLRSPNNFL SYYRLTRFLS RVIKCDPDCL RACQEQIEAL
301 LESSLRQAQQ NMDPKAAEEE EEEEEEEEVD LACTPTDVRD VDIASMGGGS
351 GGGSGGGSGG GSGGGSGGGS GLSSKGGGGS GGGGSGGGGS MATSRYEPVA
401 EIGVGAYGTV YKARDPHSGH FVALKSVRVP NGGGGGGGLP ISTVREVALL
451 RRLEAFEHPN WRLMDVCAT SRTDREIKVT LVFEHVDQDL RTYLDKAPPP
501 GLPAETIKDL MRQFLRGLDF LHANCIVHRD LKPENILVTS GGTVKLADFG
551 LARIYSYQMA LTPVWTLWY RAPEVLLQST YATPVDMWSV GCIFAEMFRR
601 KPLFCGNSEA DQLGKIFDLI GLPPEDDWPR DVSLPRGAFP PRGPRPVQSV
651 VPEMEESGAQ LLLEMLTFNP HKRISAFRAL QHSYLHKDEG NPE
Skilled artisans will recognize that the proteins of the present invention can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis, or recombinant methods. Both methods are described in U.S. Patent 4,617,149, herein incorporated by reference.
The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e.g. , H. Dugas and C. Penney, BIOORGANIC CHEMISTRY, (1981) Springer-Verlag, New York, pgs . 54- 92. For examples, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t- butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.
Sequential t-butoxycarbonyl chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides . For the production of C-terminal acids , the corresponding pyridine-2-aldoxime methiodide resm is used. Asparagine, glutamine, and arginine are coupled
preformed hydroxy benzotriazoie esters. The following side chain protection may be used: Arg, Tosyl Asp, cyclohexyl Glu, cyclohexyl Ser, Benzyl
Thr, Benzyl Tyr, 4-bromo carbobenzoxy
Removal of the t-butoxycarbonyl moiety (deprotection) may be accomplished with trifluoroacetic acid (TFA) in methylene chloride. Following completion of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees centigrade or below, preferably -20*C for thirty minutes followed by thirty minutes at 0"C. After removal of the hydrogen fluoride, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and then lyophilized. Purification is accomplished by size-exclusion chromatography on a Sephadex G-10 (Pharmacia) column in 10% acetic acid. The proteins of the present invention may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired. A general method for the construction of any desired DNA sequence is provided in J. Brown, et al.. Methods in Enzvmolocrv. 68:109 (1979) . See also, J. Sambrook, et al.. supra.
The basic steps in the recombinant production of desired proteins are:
a) construction of a synthetic or semi- synthetic DNA encoding the protein of interest;
b) integrating said DNA into an expression vector in a manner suitable for the expression of the protein of interest, either alone or as a fusion protein;
c) transforming an appropriate eukaryotic or prokaryotic host cell with said expression vector,
d) culturing said transformed or transfected host cell in a manner to express the protein of interest; and
e) recovering and purifying the recombinantly produced protein of interest.
In general, prokaryotes are used for cloning of DNA sequences in constructing the vectors of this invention. Prokaryotes may also be employed in the production of the protein of interest. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. A commercially available E. coli strain which is preferred for prokaryotic expression of the fusion proteins of the invention is designated DH10B. DH10B is available from Gibco BRL, P.O. Box 68, Grand Island, N.Y. 14072-0068. Other strains of E. coli which may be used (and their relevant genotypes) include the following.
Strain Genotype
DH5a F~ (φ80dlacZDM15) , D(lacZYA-argF)U169 supE44, hsdR17(rκ-/ mκ+) , recAl, endAl, gyrA96, thi-1, relAl HB101 supE44, hsdS20(rB~ IT©-) ' recA13, ara-14, proA2 lacYl, galK2, rpsL20, xyl-5, mtl-1, mcrB, mrr JM109 recAl, el4~(mcrA), supE44, endAl, hsdRl7(rκ~, mκ+), gyrA96, relAl, thi-1, Δ(lac-proAB) ,
F' [traD36, proAB+ lacl^, lacZΔMlδ] RR1 supE44, hsdS20(rB~ ΠIB") , ara-14 proA2, lacYl, galK2, rpsL20, xyl-5, mtl-5
chil776 F", ton, A53, dapD8, minAl, supE42 (glnV42),
D(gal-uvrB)40, minB2, rfb-2, gyrA25, thyAl42, oms- 2 , metC65, oms-1, D(bioH-asd) 29, cycB2, cycAl, hsdR2
294 endA, thi~, hsr~, hsmk+ (U.S. Patent 4,366,246)
LE392 F-, hsdR514 (r-m-), supE44, supF58, lacYl, galK2, galT22, metBl, trpR55
These strains are all commercially available from suppliers such as: Bethesda Research Laboratories, Gaithersburg, Maryland 20877 and Stratagene Cloning Systems, La Jolla, California 92037; or are readily available to the public from sources such as the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland, 10852- 1776.
Except where otherwise noted, these bacterial strains can be used interchangeably. The genotypes listed are illustrative of many of the desired characteristics for choosing a bacterial host and are not meant to limit the invention in any way. The genotype designations are in accordance with standard nomenclature. See, for example, J. Sambrook, et al. , supra. A preferred strain of E. coli employed in the cloning and expression of the genes of this invention is RV308, which is available from the ATCC under accession number ATCC 31608, and is described in United States Patent 4,551,433, issued November 5, 1985. The three E. coli host cells transformed with the vectors described in Figures 1,2 and 3 and discussed in preceding sections will be publicly available upon issuance of a patent in a "Budapest Treaty" country and thus are the preferred means for prokaryotic expression of the fusion proteins which are described herein as illustrative of the fusion proteins of the invention. The fusion proteins produced by the E. coli "deposits" of the invention require solubilization, folding and phosphorylation for complete biological activity. While they are still preferred when substantial amounts of fusion protein are desired, the facile nature of numerous eukaryotic expression systems results in a preference for these systems when modest amounts of the biologically active fusion proteins are desired.
In addition to the strains of E. coli discussed supra. bacilli such as Bacillus subtilis. other enterobacteriaceae such as Salmonella tvphimuriu or Serratia marcescans, and various Pseudomonas species may also be used. In addition to these gram-negative bacteria, other bacteria, especially Streptomvces, spp., may be employed in the prokaryotic cloning and expression of the proteins of this invention.
Promoters suitable for use with prokaryotic hosts include the b-lactamase [vector pGX2907 (ATCC 39344) contains the replicon and b-lactamase gene] and lactose promoter systems [Chang £L_al. , Nature (London) . 275:615 (1978) ; and Goeddel e _ai., Nature (London) . 281:544 (1979)] , alkaline phosphatase, the tryptophan (trp) promoter system [vector pATHl (ATCC 37695) is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter] and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) . However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems will also contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.
The proteins of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired peptide, or provides a convenient means of purifying the protein of interest. A variety of peptidases (e.g. trypsin) which cleave a polypeptide at specific sites or digest the peptides from the
amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g. , P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13 in PROTEIN
PURIFICATION: FROM MOLECULAR MECHANISMS TO LARGE SCALE PROCESSES,
American Chemical Society, Washington, D.C. (1990).
In addition to cloning and expressing the genes of interest in the prokaryotic systems discussed above, the proteins of the present invention may also be produced in eukaryotic systems. The present invention is not limited to use in a particular eukaryotic host cell. A variety of eukaryotic host cells are available from depositories such as the American Type Culture Collection (ATCC) and are suitable for use with the vectors of the present invention. The choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the cyclin-CDK fusion protein-encoding nucleic acids of the present invention. Exemplary host cells suitable for use in the present invention are listed in Table I
Table I
Host Cell Origin Source
HepG-2 Human Liver Hepatoblasto a ATCC HB 8065
CV-1 African Green Monkey Kidney ATCC CCL 70
LLC-MK2 Rhesus Monkey Kidney ATCC CCL 7
3T3 Mouse Embryo Fibroblasts ATCC CCL 92
CHO-K1 Chinese Hamster Ovary ATCC CCL 61
HeLa Human Cervix Epitheloid ATCC CCL 2
RPMI8226 Human Myeloma ATCC CCL 155
H4IIEC3 Rat Hepatoma ATCC CCL 1600
C127I Mouse Fibroblast ATCC CCL 1616
293 Human Embyronal Kidney ATCC CRL 1573
HS-Sultan Human Plasma Cell ATCC CCL 1484 Plasmocytoma
BHK-21 Baby Hamster Kidney ATCC CCL 10
A preferred eukaryotic cell line of use in expressing the fusion proteins of this invention is the widely available cell line AV12-664 (hereinafter "AV12"). This cell line is available from the American Type Culture Collection under the accession number ATCC CRL 9595. The AV12 cell line was constructed by injecting a Syrian hamster in the scruff of the neck with human adenovirus 12 and isolating cells from the resulting tumor. A wide variety of vectors, some of which are discussed below, exists for the transformation of such mammalian host cells, but the specific vectors described herein are in no way intended to limit the scope of the present invention. The sequences encoding the illustrative fusion proteins of the invention are easily removed from the deposited E. coli strains by reference to the Finαres for selection of the appropriate restriction endonucleases and inserted in any of the vectors described herein through routine purification, ligation and transfection techniques. The pSV2-type vectors comprise segments of the simian virus 40 (SV40) genome that constitute a defined eukaryotic transcription unit-promoter, intervening sequence, and polyadenylation site. In the absence of the SV40 T antigen, the plasmid pSV2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal DNA. A large number of plasmid pSV2-type vectors have been constructed, such as plasmid pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg, and pSV2-b-globin, in which the SV40 promoter drives transcription of an inserted gene. These vectors are suitable for use with the coding sequences of the present invention and are widely available from
sources such as the ATCC or the Northern Regional Research Laboratory (NRRL), 1815 N. University Street, Peoria, Illinois, 61604.
The plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification can result in the amplification of closely-associated DNA sequences and can, therefore, be used to increase production of a protein of interest. See, e.g.. R. T. Schimke, Cell,
35:705-713 (1984) .
Plasmids constructed for expression of the proteins of the present invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters. The present invention is in no way limited to the use of the particular promoters exemplified herein. Promoters such as the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene, and the major early and late adenovirus genes can be readily isolated and modified to express the genes of the present invention. Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of this invention. Furthermore, a large number of retroviruses are known that infect a wide range of eukaryotic host cells. The long terminal repeats in the retroviral DNA frequently encode functional promoters and, therefore, may be used to drive expression of the nucleic acids of the present invention.
Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman, et al. ,
Proceedings of the National Academy of Sciences (USA) , 79:6777 (1982). The plasmid pMSVi (NRRL B-15929) comprises
-34-
the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells. The mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the expression of the nucleic acids of the present invention.
The mouse metallothionein promoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which can serve as the starting material of other plasmids of the present invention.
An especially useful expression vector system employs one of a series of vectors containing the BK enhancer, an enhancer derived from the BK virus, a human papovavirus. The most preferred such vector systems are those which employ not only the BK enhancer but also the adenovirus-2-early region IA (ElA) gene product. The ElA gene product (actually, the ElA gene produces twc products, which are collectively referred to herein as "the ElA gene product") is an immediate-early gene product of adenovirus, a large DNA virus.
A preferred eukaryotic expression vectcr employed in the present invention is the phd series of vectors which comprise a BK enhancer in tandem with the adenovirus late promoter to drive expression of useful products in eukaryotic host cells. The construction and method of using the phd plasmid, as well as related plasmids, are described in U.S. Patents 5,242,688, issued September 7, 1993, and 4,992,373, issued February 12, 1991, all of which are herein incorporated by reference. Escherichia coli K12 GM48 cells harboring the plasmid phd are available as part of the permanent stock collection of the Northern Regional Research Laboratory under accession number NRRL B-18525. The plasmid may be isolated from this culture using standard techniques. The plasmid phd contains a unique Bell site which may be utilized for the insertion of the gene encoding the protein of interest. The skilled artisan understands that linkers or adapters may be employed in cloning the gene of interest into this Bell site. The phd series of plasmids functions most efficiently when introduced into a host cell
which produces the ElA gene product, cell lines such as AV12- 664, 293 cells, and others, described supra.
Transformation of the mammalian cells can be performed by any of the known processes including, but not limited to, the protoplast fusion method, the calcium phosphate co-precipitation method, electroporation and the like. See, e.g. , J. Sambrook, et al. , supra. at 3:16.30-
3:16.66.
Other routes of production are well known to skilled artisans. In addition to the plasmids discussed above, it is well known in the art that some viruses are also appropriate vectors. For example, the adenovirus, the adeno- associated virus, the vaccinia virus, the herpes virus, the baculovirus, and the rous sarcoma virus are useful. Such a method is described in U.S. Patent 4,775,624, herein incorporated by reference. Several alternate methods of expression are described in J. Sambrook, et al. , supra. at 16.3-17.44.
In addition to prokaryotes and mammalian host cells, eukaryotic microbes such as yeast cultures may also be used. The imperfect fungus Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces sp., the plasmid YRp7 (ATCC-40053) , for example, is commonly used. See, e.g. , L. Stinchcomb, ≤t fii. Nature (London) . 282:39 (1979); J. Kingsman e_L_al• Gene. 7:141 (1979); S. Tschemper et al. , Gene, 10:157 (1980). This plasmid already contains the trp gene which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [found on plasmid pAP12BD (ATCC 53231) and described in U.S. Patent No. 4,935,350, issued June 19, 1990, herein incorporated by reference] or other glycolytic enzymes such as enolase [found on plasmid pACl (ATCC 39532)], glyceraldehyde-3-phosphate dehydrogenase [derived from
plasmid pHcGAPCl (ATCC 57090, 57091)], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase, as well as the alcohol dehydrogenase and pyruvate decarboxylase genes of Zvmomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991, herein incorporated by reference) .
Other yeast promoters, which are inducible promoters, having the additional advantage of their transcription being controllable by varying growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein [contained on plasmid vector pCL28XhoLHBPV (ATCC 39475) and described in United States Patent No. 4,840,896, herein incorporated by reference], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose [e.g. GAL1 found on plasmid pRYl21 (ATCC 37658)] utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman e£ al., European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjuction with the CYC1 promoter on plasmid YEpsec--hIlbeta ATCC 67024) , also are advantageously used with yeast promoters.
Skilled artisans also recognize that some alterations of SEQ ID NO:2, 3, 5, 6, 7 or 8 will fail to change the function of the amino acid compound. For instance, some hydrophobic amino acids may be exchanged for other hydrophobic amino acids. Those altered amino acid compounds which confer substantially the same function in substantially the same manner as the exemplified amino acid compound are also encompassed within the present invention. Typically such conservative substitutions attempt to preserve the: (a) secondary or tertiary structure of the polypeptide backbone; (b) the charge or hydrophobicity of the residue; or
(c) the bulk of the side chain. Some examples of such conservative substitutions of amino acids, resulting in the production of proteins which are functional equivalents of the proteins of SEQ ID NO:2, 3, 5, 6, 7 or 8 are shown in
Table II, infra. Table II
These substitutions may be introduced into the protein in a variety of ways, such as during the chemical synthesis or by chemical modification of an amino acid side chain after the protein has been prepared.
Alterations of the protein having a sequence which corresponds to the sequences of SEQ ID NO:2, 3, 5, 7 or 8 may also be induced by alterations of the nucleic acid
compounds which encodes these proteins. These mutations of the nucleic acid compound may be generated by either random mutagenesis techniques, such as those techniques employing chemical mutagens, or by site-specific mutagenesis employing oligonucleotides. Those nucleic acid compounds which confer substantially the same function in substantially the same manner as the exemplified nucleic acid compounds are also encompassed within the present invention.
Other embodiments of the present invention are nucleic acid compounds which comprise isolated nucleic acid sequences which encode SEQ ID NO: 2, 3, 5, 7, and 8. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet due to the degeneracy of the amino acid code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences. The genes encoding the DNA molecules of the present invention may be produced using synthetic methodology. This synthesis of nucleic acids is well known in the cirt. See, e.g.. E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzvmologv, 68:109-151 (1979) . The DNA segments corresponding to the fusion proteins are generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) which employ phosphoramidite chemistry. In the alternative, the more traditional phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. See, e.g. , M.J. Gait, ed.,
OLIGONUCLEOTIDE SYNTHESIS , A PRACTICAL APPROACH , ( 1984 ) .
The DNA sequences of the present invention may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids.
The choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the molecule. A variety of other such cleavage sites may be incorporated depending on the particular plasmid constructs employed and may be generated by techniques well known in the art.
In an alternative methodology, the human cyclin and human CDK coding regions of the desired DNA sequences can be generated using the polymerase chain reaction as described in U.S. Patent No. 4,889,818, which is herein incorporated by reference.
The preferred expression systems for use in the present invention are the various Baculovirus systems. The pFastBacl expression system, which is commercially available from the Life Technologies group of Gibco BRL Products as
Catalog No. 10360-016. Life Technologies, P.O. Box 68, Grand Island, NY 14072, Telephone: 800 828 6686, is the preferred expression system when modest amounts of biologically active fusion proteins are desired. The Bac-To-Bac Baculovirus Expression System has been used for expression of the sequences of the present invention and this system is also available from Life Technologies (Catalog No. 10359-016). The present inventors elected to deposit the DNA sequences encoding the illustrative cyclin-CDK fusion proteins as components of prokaryotic, lac operon-regulated expression systems due to the ability of the E. coli systems to produce large amounts of the fusion proteins and the ease with which skilled artisans can excise the desired coding sequences from the E. coli systems and insert them into these commerically available Baculovirus expression systems to thereby achieve the preferred mode of expressing modest amounts of the illustrative fusion proteins.
Baculovirus expression systems are well known in the art and numerous scientific articles and "methods" books are available on the subject. The present inventors have found the Life Technologies technical literature to provide excellent guidance for producing products of interest via
-40 -
Baculovirus expression. The preferred techniques for Baculovirus expression of the sequences of the present invention are those provided in the product literature. Minor variations such as linker construction and the like are considered in light of the advanced state of this art as too trivial to warrant discussion. In the event skilled artisans elect to depart from the commercially available Baculovirus systems, the present inventors recommend Baculovirus Expression Vectors-A Laboratory Manual, O'Reilly, David R., Miller, Lois K., and Luckow, Verne A., W. H. Freeman and Company, New York, New York as a source of additional information on any protocol required for successful expression of polypeptides in Baculovirus systems.
The assays which are greatly advantaged by the fusion proteins of the present invention are well illustrated in two recent scientific publications: Connell-Crowley, L., et al . , Mol. Biol. of the Cell 4, 79-92 (1993) and Desai, D., Mol. Biol. of the Cell 3, 571-582 (1992) .
The examples provide sources for reagents, however it will be understood that numerous vendors market reagents of high quality for use in the protocols and procedures described below and the substitution of reagents or protocols is contemplated by the present invention and embraced in the scope thereof. All temperatures unless otherwise noted are expressed in degrees Centigrade. All percentages are on a weight per weight basis unless otherwise noted.
Skilled artisans wishing to practice the recombinant DNA aspects of the present invention are directed to the NIH guidelines for information on research involving recombinant DNA molecules. A copy of the current guidelines can be obtained from Office of Recombinant DNA Activities, National Institutes of Health, Building 31, Room 4B11, Bethesda, MD 20892. Compliance with all such current regulations regarding vector selection, expression of human and animal genes and containment requirements is required by law.
The examples are intended to further illustrate the present invention and are not to be interpreted as limiting on the scope thereof. While the examples and detailed description sections of the present invention are sufficient to guide anyone of ordinary skill in the art in the practice of the present invention, skilled artisans are also directed to Mol ecular Cloning A Laboratory Manual Second Edition, Sambrook,J., Fritsch, E. F., and Maniatis, T., Cold Spring Harbor Press 1989 and Current Protocols In Molecular Biology, Ausubel, F.M., Brent,R., Kingston,R.E. , Moore, D. D., Seidman,J.G. , Smith, J.A., and Struhl, K., Ed. Greene Publishing Associates and Wiley-Interscience 1989. The aforementioned resources provide an excellent technical supplement to any discourse in genetic engineering.
Example 1 Production of Baculovirus System for Expression of SEQ ID NO:2
A sample of NRRL B-21490 is obtained from the NRRL. The sample is cultured according to well known procedures using standard media containing Ampicillin for selection of the desired transformed phenotype.
Plasmid isolation is accomplished in accordance with standard methodology. See e.g. Sambrook and Maniatis, supra.
The desired fragment is excised from plasmid pK415 (See Figure 1) by sequential digestion with the restriction endonucleases, Ascl and Sse 83871. The Ascl digestion is performed using New England Biolabs reagents and protocols. The restriction endonuclease Sse 83871 is available from Takara Biomedicals via PanVera Corp., 565 Science Drive, Madison, WI 53711 (1 800 791-1400) . The vendors instructions on digestion procedures are recommended. pFastBacI is digested with BssHII (New England Biolabs) and PstI (New England Biolabs) in accordance with vendors instructions and the large fragment is isolated. A restriction site and function map of pFastBacI is provided at
page 5 of the GibcoBRL/Life Technologies Catalog Number 10359-016 (Instruction Manual-BAC-TO-BAC™ Baculovisur Expression System) . The catalog is herein incorporated by reference. The fusion protein encoding sequence is then ligated into the pFastBacl vector using standard ligation reagents and conditions. Preferred ligation reaςrents and conditions are set forth at pages 7 and 8, Section 3.3, of GibcoBRL/Life Technogies Catalog Number 10359-016. Page 5 of GibcoBRL/Life Technogies Catalog Number 10359-016 provides DNA sequence information and restriction endonuclease cleavage sites for the multiple cloning site of pFastBacl and is therefore useful in the event skilled artisans elect to fragment the sequence from p415 or excise it by other than the restriction endonucleases suggested above and utilize linkers to facilitate the subsequent ligation into pFastBacl. Transposition of the pFastBacl vector comprising the fusion protein of plasmid pK415 into DHlOBaclO (competent cells are provided as part of the expression kit accompanying pFastBacl in Catalog Number 10359-16) is conducted in accordance with the teachings of page 8 of GibcoBRL/Life Technogies Catalog Number 10359-016.
Isolation of Recombinant Bacmid DNA is accomplished in accordance with the teachings of pages 8 and 9 of GibcoBRL/Life Technogies Catalog Number 10359-016. Transfection of Sf9 cells with recombinant Bacmid DNA, harvesting and storage of the recombinant Baculovirus, and Infection of Insect Cells with recombinant Baculovirus particles is accomplished with the teachings at pages 9 and 10 of GibcoBRL/Life Technogies Catalog Number 10359-016.
Example 2 Production of Baculovirus System for Expression of SEQ ID NO:4
Baculovirus expression systems were constructed in substantial accordance with the teachings of Example 1.
Plasmid pK480 from E. coli/pK485 was used in place of plasmid
pk415 as the source of the DNA sequence encoding the fusion protein of interest.
Example 3 Production of Baculovirus System for Expression of SEQ ID NO:6
Baculovirus expression systems were constructed in substantial accordance with the teachings of Example 1. Plasmid pK485 from E. coli/pK480, NRRL number B21491, was used in place of plasmid pK415 as the source of the DNA sequence encoding the fusion protein of interest. With the exception of the substitution of plasmid pK480 for plasmid pK415 all steps of this Example 3 were carried out in conformance with the teachings of Example 1.
Example 4 Purification of Co-expressed D1.K4
Affinity chromatography resins for fusion protein purification are readily constructed from commercially available reagents using techniques well known in the art. CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) is the preferred matrix for linkage of appropriate monoclonal or polyclonal antibodies to allow antibody-based affinity purification of the fusion proteins. Pharmacia Fine Chemicals publishes "Affinity Chromatography-Principles and Methods". This manual sets forth all steps in preparing the affinity resin and performing the antibody-based affinity purification steps. The manual is available from Pharmacia Fine Chemicals, Box 175, S-751 04 Uppsala 1, Sweden.
Example 5
Strepavadin Purification of Cyclin-CDK Fusion Proteins
The SF9 cells which were utilized in Examples 1-3 as the host cells for Baculovirus expression were collected by centrifugation and resuspended and lysed via sonication at 4°C in Resuspension Buffer at a density of 8 X 10^/mL.
Resuspension buffer is 50mM HEPES pH 7.5, 0.32M Sucrose, 0.1 mM PMSF, l.OmM DTT, ImM EDTA and 80mM β-glycerophosphate.
500μL of the SF9 extract was added to 200μL of Streptavidin Paramagnetic Beads (Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399) and the mixture was incubated at room temperature for 45 minutes . The paramagnetic beads were pelleted at room temperature using a MagneSphere Technology Magnetic separation stand (Promega) . The beads were washed three times with 1 mL of 1XPBS/25 mg/ml BSA (or 0.1% Tween 20) at room temperature.
The fusion protein was eluted from the beads in 120 μL of Elution Buffer A for 30 minutes at room temperature. Elution Buffer A is 25mM HEPES pH 7.5, 0.1 mM PMSF, ImM d- Biotin O.lmM DTT, 20mM β-glycerophosphate, ImMNaF, lOmM Sodium Orthovanadate and 10% glycerol.
The purified fusion protein was stored at -70' C until ready for use.
Ni-NTA Purification of Cyclin-CDK Fusion Proteins 8 X 106 SF6 cells/mL (from Examples 1-3) were collected by centrifugation and resuspended and ÷.ysed at 4°C in Resuspension Buffer. 1.0 mL of the insect cell extract was added to 3.0 mL of Ni-NTA agarose (Qiagen Inc., 9259 Eton Avenue, Chatsworth, CA 91311), which was previously equilibrated with Wash Buffer. Wash Buffer is 50mM HEPES pH
7.5, 300 mM NaCl, 20mM Imidizole and 0.1 mM PMSF.
The extract agarose mixture was incubated at 4'C for 4 hours. The mixture was gently agitated during the incubation. The agarose was then pelleted by centrifugation at 2000xg for two minutes and then washed three times with
5.0mL of 1XPBS at 4°C with agitation. The fusion protein was eluted from the agarose in 750 μL of Elution Buffer B for 1 hour at 4°C with agitation. Elution Buffer B is 50mM HEPES pH 7.5, 300mM NaCl, 250mM Imidizole, 0.1 mM PMSF,, lOmM Sodium Orthovanadate, ImM NaF and 20mM β-glycerophosphate. The
eluted fusion protein was dialyzed in 3.0L of Dialysis Buffer overnight at 4'C. Dialysis Buffer is 25mM HEPES ph 7.5, 10% glycerol, 0.01% Triton-X, 0.ImM PMSF,20mM β-glycerophosphate, ImM NaF and lOmM Sodium Orthovanadate. The dialyzed fusion protein was stored at -70*C.
Example 7
Purification of Co-expressed D1.K4 Individual Units
Purification of co-expressed cyclin Dl and cdk4 was performed at Spinx Pharmaceuticals. Insect cell pellets were homogenized at 1:10 in 50 mM HEPES pH 7.5, 320 mM Sucrose, ImM DTT, O.lmM PMSF, ImM EGTA, ImM EDTA and 20μg/ml leupeptin. The lysed cells were spun for 1.5 hrs . at 100,000 xg to remove cytosol then equilibrated a Poros Q column in Equilibration Buffer (25mM Tris pH 8.0, 10% glycerol, ImM DTT, O.lmM PMSF, ImM EDTA, and 20 μg/ml leupeptin) . The lysates were loaded onto a Poros Q column at 5ml/L of infected insect cells. The Poros Q column was washed with 10-column volumes of Equilibration buffer. The column was eluted with 0-1M NaCl gradient collecting 2ml/fraction. The column fractions were assayed for activity and peak fractions were pooled. The resulting pool was diluted to give a final NaCl concentration of lOOmM. The dilute pool fractions were loaded onto a Hydroxapatite column equilibrated with 25mM Tris pH 8.0, 0.1 mM PMSF, ImM EDTA, and 20 μg/ml leupeptin.
The Hydroxapatite column was washed with 10-column volumes of Equilibration buffer and eluted cyclin Dl and cdk4 with 0- 400mM potassium phosphate, pH 7.5. Column fractions were assayed for activity and the peak fractions pooled. The eluted protein was stored at -70C.
Immunoprecipitation of D1.K4 Fusion
5 x 106 cells/mL were lysed in IP Lysis Buffer on ice for 30 minutes (IP Lysis Buffer: 50mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, ImM DTT, 2.5mM EGTA, 0.1% Tween 20, 10%
Glycerol, O.lmM PMSF, 500UM ATP, lOmM β-glycerophosphate, ImM NaF, and O.lmM orthovanadate) . The cells were sonicated three times on ice for 10 seconds each time, and the lysates were clarified for 5 minutes at 10,000 rpm and °C. 20μL of myc antibody (lOOμg/mL commercially available from Oncogene Science, Cambridge, Mass.) was added to 500μL of clarified cell lysate. The mixture was incubated with agitation for 3 hours at 4°C. 50μl of 50% Protein-G Agarose (Boehringer Mannheim) , which had been washed with IP Lysis Buffer, was then added to each sample. The samples were incubated with agitation for 2-5 hours at 4°C. The Protein-G-Agarose was pelleted and washed 4X with IP Lysis Buffer and then 2X with 50mM HEPES pH 7.4 and ImM DTT. The washed Protein-G-Agarose was resuspended in Kinase Reaction Buffer.
Example 9
Assays for Clyclin Dl and cdk4
Partially purified co-expressed or fused cyclin Dl and cdk4 were assayed for Rb kinase activity. Co-expressed cyclin Dl and cdk4 were partially purified as described above. Fused cyclin Dl-cdk4 was partially purified by streptavidin beads, Ni-NTA agarose, and by immunoprecipitation. In immunoprecipitations, fused cyclin Dl-cdk4 expressed in stably transfected Rat Embryo Fibroblasts (E3600NA-FPr-5) were partially purified as described in Matsushime et al.. 1994. Kinase redactions with various amounts of partially purified cyclin Dl and cdk4 from insect cells contained: 50mM HEPES pH 7.5, lOmM MgCl2, 0.2μCi [gamma-32 P]ATP (Amersham, 6,000 Ci/mmol) , 0.12μg pRb (full-length protein from Immuno Pharmaceutics), O.lmM sodium orthovanadate, lOmM β-glycerophophate and ImM NaF in a total of 100μL. Kinase reactions with immunoprecipitated fusion protein on Protein-G-Agarose (Boehringer Mannheim) from the REF cell line were resuspended in 50 μl of Kinase Reaction Buffer (50mM HEPES pH 7.5, lOmM MgCl2, lO.OμCi | gamma-32p]ATP (Amersham, 6,000 Ci/mmol) , 0.2μg pRb (full-length protein from Immuno Pharmaceutics), ImM DTT, 2.5 mM EGTA, 20μM ATP,
O.lmM sodium orthovanadate, lOmM β-clycerophophate and ImM NaF) . Reactions were incubated at 30°C for 30 minutes, boiled for 5 minutes, and half of the reaction was loaded onto a 12.5% SDS-polyarcylamide gel. The gel was transferred to Hybond-ECL nitrocellulose (Amersham) and exposed to Hyperfilm-ECL (Amersham) .
Example 10
Immunoblots
For protein detection of cyclin Dl and cdk4, nitrocellulose membranes were blocked with 5% dry milk in 1 x PBS for 30 to 60 minutes. Membranes were washed 3x, 10 minutes for each wash, in lx PBS/0.1% Tween 20. The membrane was incubated with primary antibody (cyclin Dl or cdk4) at a 1:2000 dilution in IX PBS/0.1% Tween 20/1% Milk for 1 hour at room temperature then washed 3X for 10 minutes each in IX PBS/0.1% Tween 20. The membrane was then incubated with a secondary antibody (horse radish peroxidase conjugated goat anti-mouse or rabbit antibody from Amersham) at a 1:1000 dilution in IX PBS/0.1% Tween 20/1% Milk for 25 minutes at room temperature. The membrane was washed 6X in PBS/0.1% Tween 20, 2X in IX PBS, and developed with Amersham ECL detection reagents. The results indicated that the fusion protein had substantially the same amount of activity as the individual subunits.
Claims
1. A fusion protein comprising a human cyclin and a human CDK.
2. The fusion protein of Claim 1 wherein one or more of the amino acid residues of said human cyclin are replaced by conservative substituions.
3. The fusion protein of Claim 1 wherein one or more of the amino acid residues of said human CDK are replaced by conservative amino acid substituions .
4. The fusion protein of Claim 1 wherein said human cyclin in human cyclin Dl .
5. The fusion protein of Claim 1 wherein said human CDK is human CDK4.
6. The fusion protein of Claim 1 that is SEQ ID NO:2.
7. The fusion protein of Claim 1 that is SEQ ID NO:3.9
8. The fusion protein of Claim 1 that is SEQ ID NO:5.
9. The fusion protein of Claim 1 that is SEQ ID No. 7
8.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU15268/97A AU1526897A (en) | 1996-01-05 | 1997-01-03 | Fusion proteins comprising cell cycle regulatory proteins |
| EP97901350A EP0871659A4 (en) | 1996-01-05 | 1997-01-03 | Fusion proteins comprising cell cycle regulatory proteins |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US962996P | 1996-01-05 | 1996-01-05 | |
| US60/009,629 | 1996-01-05 |
Publications (1)
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|---|---|
| WO1997025345A1 true WO1997025345A1 (en) | 1997-07-17 |
Family
ID=21738815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/000140 WO1997025345A1 (en) | 1996-01-05 | 1997-01-03 | Fusion proteins comprising cell cycle regulatory proteins |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0871659A4 (en) |
| AU (1) | AU1526897A (en) |
| CA (1) | CA2241849A1 (en) |
| WO (1) | WO1997025345A1 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999025829A2 (en) * | 1997-11-13 | 1999-05-27 | Curagen Corporation | Cdk2 protein and cdk2 protein complexes |
| WO1999033962A1 (en) * | 1997-12-30 | 1999-07-08 | Chiron Corporation | HUMAN CYCLIN-DEPENDENT KINASE (hPFTAIRE) |
| KR20000019889A (en) * | 1998-09-16 | 2000-04-15 | 성재갑 | Preparation method for cdk4/p16 complex using insect cells |
| WO2000036118A2 (en) * | 1998-12-16 | 2000-06-22 | Chiron Corporation | HUMAN CYCLIN-DEPENDENT KINASE (hPNQALRE) |
| WO2002077018A1 (en) * | 2001-03-21 | 2002-10-03 | Iba Gmbh | Sequentially arranged streptavidin-binding modules as affinity tags |
| US6716626B1 (en) | 1999-11-18 | 2004-04-06 | Chiron Corporation | Human FGF-21 nucleic acids |
| EP2075255A1 (en) | 2000-03-08 | 2009-07-01 | Novartis Vaccines and Diagnostics, Inc. | Human FGF-23 gene and gene expression products |
| EP2163626A1 (en) | 1999-11-18 | 2010-03-17 | Novartis Vaccines and Diagnostics, Inc. | Human FGF-21 gene and gene expression products |
| WO2014037460A3 (en) * | 2012-09-05 | 2014-05-30 | Universite Libre De Bruxelles | Immobilised cyclin-dependent kinase 4 fusion proteins and uses thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5338669A (en) * | 1989-09-20 | 1994-08-16 | Abbott Biotech, Incorporated | Method of producing fusion proteins |
-
1997
- 1997-01-03 EP EP97901350A patent/EP0871659A4/en not_active Withdrawn
- 1997-01-03 CA CA 2241849 patent/CA2241849A1/en not_active Abandoned
- 1997-01-03 AU AU15268/97A patent/AU1526897A/en not_active Abandoned
- 1997-01-03 WO PCT/US1997/000140 patent/WO1997025345A1/en not_active Application Discontinuation
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|---|---|---|---|---|
| WO1999025829A3 (en) * | 1997-11-13 | 1999-09-10 | Curagen Corp | Cdk2 protein and cdk2 protein complexes |
| US5986055A (en) * | 1997-11-13 | 1999-11-16 | Curagen Corporation | CDK2 interactions |
| US6521412B1 (en) | 1997-11-13 | 2003-02-18 | Curagen Corporation | HsReq*1 and hsReq*2proteins and use thereof to detect CDK2 |
| WO1999025829A2 (en) * | 1997-11-13 | 1999-05-27 | Curagen Corporation | Cdk2 protein and cdk2 protein complexes |
| WO1999033962A1 (en) * | 1997-12-30 | 1999-07-08 | Chiron Corporation | HUMAN CYCLIN-DEPENDENT KINASE (hPFTAIRE) |
| US6432668B1 (en) | 1997-12-30 | 2002-08-13 | Chiron Corporation | Polynucleotides encoding human cyclin-dependent kinase (hPFTAIRE) |
| KR20000019889A (en) * | 1998-09-16 | 2000-04-15 | 성재갑 | Preparation method for cdk4/p16 complex using insect cells |
| US7566559B2 (en) | 1998-12-16 | 2009-07-28 | Novartis Vaccines And Diagnostics, Inc. | Human cyclin-dependent kinase (hPNQALRE) |
| WO2000036118A2 (en) * | 1998-12-16 | 2000-06-22 | Chiron Corporation | HUMAN CYCLIN-DEPENDENT KINASE (hPNQALRE) |
| WO2000036118A3 (en) * | 1998-12-16 | 2000-11-16 | Chiron Corp | HUMAN CYCLIN-DEPENDENT KINASE (hPNQALRE) |
| US7855058B2 (en) | 1998-12-16 | 2010-12-21 | Novartis Vaccines And Diagnostics, Inc. | Human cyclin-dependent kinase (hPNQALRE) |
| US7250256B2 (en) | 1998-12-16 | 2007-07-31 | Chiron Corporation | Human cyclin-dependent kinase (hPNQALRE) |
| US6716626B1 (en) | 1999-11-18 | 2004-04-06 | Chiron Corporation | Human FGF-21 nucleic acids |
| US7259248B2 (en) | 1999-11-18 | 2007-08-21 | Chiron Corporation | Human FGF-21 polypeptides |
| EP2163626A1 (en) | 1999-11-18 | 2010-03-17 | Novartis Vaccines and Diagnostics, Inc. | Human FGF-21 gene and gene expression products |
| US7723297B2 (en) | 1999-11-18 | 2010-05-25 | Kyoto University | Human FGF-21 gene and gene expression products |
| EP2075255A1 (en) | 2000-03-08 | 2009-07-01 | Novartis Vaccines and Diagnostics, Inc. | Human FGF-23 gene and gene expression products |
| WO2002077018A1 (en) * | 2001-03-21 | 2002-10-03 | Iba Gmbh | Sequentially arranged streptavidin-binding modules as affinity tags |
| US7981632B2 (en) | 2001-03-21 | 2011-07-19 | Iba Gmbh | Sequentially arranged streptavidin-binding modules as affinity tags |
| US8735540B2 (en) | 2001-03-21 | 2014-05-27 | Iba Gmbh | Peptides with sequentially arranged streptavidin binding modules |
| WO2014037460A3 (en) * | 2012-09-05 | 2014-05-30 | Universite Libre De Bruxelles | Immobilised cyclin-dependent kinase 4 fusion proteins and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU1526897A (en) | 1997-08-01 |
| EP0871659A4 (en) | 2000-04-26 |
| EP0871659A1 (en) | 1998-10-21 |
| CA2241849A1 (en) | 1997-07-17 |
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